Role of the Carbohydrate Moiety in Phospholipase B from Torulaspora delbrueckii

Agric. Bioi Chern., 54 (3), 599-603, 1990 599 Role of the Carbohydrate Moiety in Phospholipase B from Torulaspora delbrueckii Masafumi Maruyama, Hideki Kadowaki, Yasuo Watanabe and Youichi Tamai Department of Bioresources, Faculty of Agriculture, Ehime University, Matsuyama, Ehime 790, Japan Received July 31, 1989 Water-soluble phospholipase B containing approximately 50 % carbohydrate, purified to homogeneity from yeast cell washings of Torulaspora delbrueckii, was treated with endoglycosidase H (Endo-H), and about 75 %of the carbohydrate associated with this enzyme was removed. The native and carbohydrate-depleted enzymes were compared. The phospholipase B activity was higher in the carbohydrate-depleted enzyme than in the native form. The enzymatic activation by the removal of carbohydrate was due to the increased affinity of the enzyme for the substrate. The carbohydratedepleted phospholipase B was less stable upon incubation at 50 C and more susceptible to proteolysis by V8 protease. These results suggested that the carbohydrate of phospholipase B in yeast cells stabilizes the enzyme conformation and protects the enzyme from proteolysis. The biological significance of the carbohydrate moiety of glycoprotein has been suggested in several enzymes.1~5) Among them, cell wall invertase5) and phospholipase B6~9) are typical large carbohydrate-containing enzymes in yeast cells. The roles of the carbohydrate moieties in yeast cell wall invertase have been investigated by Frederick et al.3) and Yamamotoet alp and several functions have been suggested. However, few studies have been done on the role of carbohydrate associated with phospholipase B in yeast. Recently, we isolated two types of phospholipase B, membrane-bound and water-soluble phospholipase B, from the yeast Torulaspora delbrueckii.6'1) Both types of enzymes were glycoproteins containing approximately 50% carbohydrate. These yeast phospholipase B were significantly activated at softening in storage, and were thought to be important in the autolysis process of yeast cells. But, in the process of autolysis, concomitant proteolytic enzyme activation has been also observed.10) These facts suggested that phospholipase B activated at softening might be resistant to proteolytic degradation. In this paper, the properties of native and carbohydrate-deplet- ed forms of phospholipase B were compared and found to differ considerably, not only in stability to proteolytic enzyme or heating treatment, but also in kinetic properties of enzyme activity. Materials and Methods Materials. Water-soluble phospholipase B was purified from T. delbrueckii cell washings by sequential column chromatographies on Octyl-Sepharose CL-4B, DEAE- Sephacel, and Sepharose 6B as described in our previous paper.6) The purified enzyme gave a broad stained band on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Phosphatidylcholine and V8 protease were purchased from Sigma Chemical Co. Endo-H (endo-/?-vvaeetylglucosaminidase H from Streptomyces lividans) was purchased from Boehringer Manheim. All other chemicals were of the purest grade available. Assay ofphospholipase activity. Water-soluble phospholipase B activity was assayed by the method described previously,6) The unit of activity is defined as the amount of enzyme which liberates 1 fimo\ of fatty acid per min from phosphatidylcholine at 30 C at two phs (ph 2.5 and ph 7.5). Digestion with Endo-H and isolation of the digestedphospholipase B. The purified phospholipase B (100 /ig) was incubated in 0.1 ml of0.05m citrate buffer, ph 5.5, contain-

600 M. Maruyama et al. ing 20m units ofendo-h and 1 mmpmsf(phenylmethylsulfonylfluoride) at 37 C for 24hr. To remove the carbohydrate from the phospholipase B after treatment with endo-h, the digests were put on a TSK gel G3000 SWcolumn of high pressure liquid chromatography which had been pre-equilibrated with 0.05 m Tris-HCl buffer, ph 8.0, and the treated phospholipase B was recovered. The sugar was measured by the phenol-sulfuric acid method13) using glucose as the standard. The carbohydrate removedwas calculated from the carbohydrate content of the Endo-H treated phospholipase B. Proteolytic digestion of phospholipase B. Native or Endo-H-treated phospholipase B (10/ig) was dissolved in 50^1 of 0.05m Tris-HCl buffer, ph 8.0, containing 0.1 fig of V8 protease. After incubation at 37 C for 0 to 4hr, samples (10//I) were withdrawn for analysis by SDSpolyacrylamide gel electrophoresis. Polyaerylamide gel electrophoresis. SDS-polyacrylamide slab gel electrophoresis was done with 7.5% and 10% acrylamide gel by the procedure oflaemmli.x l] The following proteins were used as molecular weight markers: carbonicanhydrase(mw29,000), ovalbumin(mw45,000), bovine serum albumin (MW66,000), phosphorylase b (MW 97,400), ^-galactosidase (MW 1 16,000), and myosin (MW 205,000). Gels were stained with Coomassie Brilliant Blue R-250. Other measurements. Protein was measured by the method of Lowry et al.l2) using bovine serum albumin as the standard. Results and Discussion Endo-H-treated phospholipase B Purified water-soluble phospholipase B was shown to be a glycoprotein containing approximately 50% carbohydrate.6* The native phospholipase B gave a protein band corresponding to a molecular weight from 170,000 to 200,000. Endo-H liberated about 75%of the total carbohydrate from the native form of phospholipase B upon incubation for 24hr. It showed a decrease in molecular weight, due to the removal of sugar chains, and three main protein bands appeared in SDS-polyacrylamide gel. No more carbohydrate was removed even when the digestion time was prolonged. But when native phospholipase B was denatured in 0.15% SDS at 100 C for 5min, and then digested with Endo-H, it showed a single band correspond- Fig. 1. SDS-Polyacrylamide Gel Electrophoresis of Native and Carbohydrate-depleted The experimental conditions are described in Materials and Methods with 7.5% polyacrylamide. Standard molecular weight marker proteins were rabbit muscle myosin (MW 205,000), /?-galactosidase (MW 116,000), rabbit muscle phosphorylase b (MW 97,400), bovine serum albumin (MW 66,000), egg albumin (MW. 45,000), and carbonic anhydrase (MW 29,000). Lane A: native phospholipase B, Lane B: Endo-H-treated phospholipase B, Lane C: phospholipase B treated with Endo-H after denaturing in the presence of 0.15% SDS. ing to MW 72,000 (Fig. 1) and the carbohydrate content of the enzyme was about 10%. These results suggest that some carbohydrate-protein linkage regions may be embedded in the interior of the enzyme molxule. Enzymatic activities of the native and carbohydrate-depleted phospholipase B The enzymatic activity of the Endo-Htreated phospholipase B was compared with that of the native enzyme. Phospholipase B from T. delbrueckii has two ph optima, one acidic (ph 2.5-3.0) and the other alkaline (ph 7.2-8.0) as reported previously.6>7) The removal of the carbohydrate moiety by Endo-H increased the activities of phospholipase B at both ph regions. However, the increased ratio was higher in the alkaline activity (Table I). Frederick et al.3) reported that yeast invertase, a representative yeast glycoprotein containing

Table I. Specific Activities of Native and Carbohydtate-depleted Phospholipase B (units/mg Role of Carbohydrate in Phospholipase B 601 Phospholipase protein) activity Acidic side Alkaline sidt Native phospholipase B 4.88 0.94 Carbohydrate-depleted phospholipase B 1 1.84 4.66 a large amount of carbohydrate, did not differ significantly in enzyme activities between native invertase and carbohydrate-depleted invertase. On the other hand, in the case oi phospholipase B from Penicillium notatum,14 the enzymatic activity was increased by the removal of 26-27% of the carbohydrate associated with the enzyme. These different results on the effects of carbohydrate might be because the substrate for invertase is easily accessible to the catalytic site of the enzyme molecule without suffering interference by the carbohydrate moiety associated with the enzyme, since sucrose, a substrate for invertase, is a small molecule. Contrary to this, phosphatidylcholine, a substrate for phospholipase B, usually exists as micel, which is a high molecular weight substance like protein, in aqueous solution. Consequently, native phospholipase B might usually suffer some interference in the formation of the enzyme-substrate complex from the carbohydrate moiety associated with the enzyme. Therefore, the activation of phospholipase B due to the removal of the carbohydrate moiety seems to be characteristic of phospholipase B from micro-organisms. Kinetic properties of native and carbohydratedepleted phospholipase B As shown in Table I, alkaline side phospholipase activity was increased more significantly by the removal of the carbohydrate moiety. In :his study, the Michaelis constant (Km) and naximal velocity (Fmax) values of native and :arbohydrate-depleted phospholipase B activities in alkaline ph were compared. The enzymereaction was done in a solution con- Fig. 2. Double-reciprocal Plots for Native and Carbohydrate-depleted The enzymeactivities were measured at acidic and alkaline phs by the method described in Materials and Methods except that the molar ratio of Triton X-100 to phosphatidylcholine in the reaction solution was maintained at 3.1 : 1. -O-, native phospholipase B activity at alkaline ph; -A-, native phospholipase B activity at acidic ph; -#-, carbohydrate-depleted phospholipase B activity at alkaline ph; -A-, carbohydrate-deple phospholipase B activity at acidic ph. taining various concentrations of Triton X- 100 which were raised in proportion to that of phosphoatidylcholine as described by Ichimasa et al}5) The molar ratio of Triton X- 100 to the phosphatidylcholine in the reaction solution was 3.1 :1. Under these conditions, a Michaelis-Menten saturation curve was obtained. Figure 2 shows the double reciprocal plots for the native and carbohydratedepleted phospholipases B. The Kmand Fmax of native phospholipase B and carbohydratedepleted phospholipase B at the alkaline side were lommand 35.7 units/mg protein, and 1.25mM and 31.7 units/mg protein, respectively. Thus, the Kmof carbohydrate-depleted phospholipase B was lower than that of the native enzyme. These results suggest that the activation is due to the fact that phosphatidyl-

602 M. Maruyama et al. choline, a substrate of phospholipase B, becomes easily accessible to the catalytic site of the enzyme, as inferred above. Thermalstabilities of native and carbohydratedepleted phosphipase B The carbohydrate-depleted enzyme was less stable to heating treatment. When carbohydrate-depleted phospholipase B was heated at 50 C for lomin, acidic and alkaline activities were decreased to 20% and 10%, respectively, while the native phospholipase B retained almost full activity at both phs at this temperature (Fig. 3). The result suggests that the carbohydrate stabilizes the configuration of phospholipase B. The same effect was observed with the carbohydrate moiety of yeast cell wall invertase16) and porcine ribonuclease.17) Effects of V8 protease on the activities of the native and carbohydrate-depleted phospholipase B To compare the susceptibilities of native and carbohydrate-depleted phospholipases B (riot denatured by SDS) to proteolytic enzymes, both forms of enzymes were digested with V8 protease, which is a highly selective proteolytic enzyme, and their phospholipase B activities were assayed at several intervals of incubation. Figure 4 shows that the carbohydrate-depleted phospholipase B was rapidly inactivated to 15% of the original activity after 3 hr of digestion. The reason why the inactivation of the alkaline side activity was more rapid than that ofacidic side activity is unknown. On the other hand, the native phospholipase B activity was more stable than its carbohydrate-depleted form under the same digestion conditions. The proteolytic degradation of both forms of phospholipase B was analyzed by SDSpolyacrylamide slab gel electrophoresis. Figure 5 shows the results. The native phospholipase B was more resistant to degradation by V8 protease, but the carbohydrate-depleted phospholipase B gave small polypeptides corresponding to molecular weights from 20,000 to 70,000 other than high molecular weight polypeptides (70,000-90,000). Thus a protective role of carbohydrate against proteolytic degradation was suggested. This protection by carbohydrate of the activity of the phospholipase B seems to be important for maintaining Fig. 3. Thermalstabilities of Native and Carbohydratedepleted The purified native and carbohydrate-depleted enzymes (each 10^g) were kept for lomin at various temperatures in 50mM Tris-HCl buffer, ph 8.0, and the remaining activities were measured by the method described in Materials and Methods. The activities are expressed as percentages of the activity in the undigested control. The symbols are the same as those for Fig. 1. Fig. 4. Effect of V8 Protease Digestion on Native and Carbohydrate-depleted The purified native and carbohydrate-depleted enzymes (each 10/xg) were digested by V8 protease (0.1 fig) in 50/^1 of 50mMTris-HCl buffer, ph 8.0. The symbols are the same as those for Fig. 1.

Role of Carbohydrate in Phospholipase B 603 Fig. 5. SDS-Polyacrylamide Gel Eectrophoresis of V8 Protease Digestion on Native and Carbohydratedepleted The experimental conditions are described in Materials and Methods with 10%polyacrylamide. Lanes: A, B, C, D, and E, native phospholipase B digested by V8 protease for Omin, 30min, 1 hr, 2hr, and 3hr, respectively, and F, G, H, I, and J Endo-H-treated phospholipase B digested for the same intervals. enzymeactivity, because yeast cells in the stage of softening also contain high protease activity besides phospholipase B activity, and activation of both enzymes are considered to be one of the main factors for inducing autolysis of yeast cells. Accordingly, we would like to conclude that the carbohydrate moiety of phospholipase B plays a role in the resistance to inactivation by yeast cytoplasmic proteases. References 1) K. Yamamoto, K. Takegawa and H. Kumagai, Agric. Biol. Chem., 51, 1481 (1987). 2) J.A. Brown,H. L. Sega, F. Maley, R. B.Trimbleand F. Chu, J. Biol. Chem., 254, 3689 (1979). 3) K. C. Frederick, R. B. Trimble and F. Maley, /. Biol. Chem., 253, 8691 (1978). 4) U. A. Reddy, R. S. Johnson, K. Biemann, R. S. Williams, F. D. Zieglar, R. B. Trimble and F. Maley, J. Biol. Chem., 263, 6978 (1988). 5) R. B. Trimble and F. Maley, J. Biol. Chem., 252, 4409 (1977). 6) Y. Kuwabara, M. Maruyama, Y. Watanabe, S. Tanaka, M. Takakuwa and Y. Tamai, Agric. Biol. Chem., 52, 2451 (1988). 7) Y. Kuwabara, M. Maruyama, Y. Watanabe, S. Tanaka, M. Takakuwa and Y. Tamai, /. Biochem., 104, 236 (1988). 8) W. Witt, M. E. Schweingruber and A. Mertsching, Biochim. Biophys. Ada, 795, 108 (1984). 9) W. Witt, A. Mertsching and E. Konig, Biochim. Biophys. Ada, 795, 117 (1984). 10) J. F. Lenney and J. M. Dalvec, Arch. Biochem. Biophys., 120, 42 (1967). ll) U. K. Laemmli, Nature, 227, 680 (1970). 12) O. H. Lowry, N.J. Rosebough,A. L. Farrand R. J. Randall, J. Biol. Chem., 193, 265 (1951). 13) M. Dubois and K. A. Gills, Anal. Chem., 28, 350 (1956). 14) J. Sugatani, T. Okumura, K. Saito, K. Ikeda and K. Hamaguchi, J. Biochem., 95, 1407 (1984). 15) M.. Ichimasa and 49, 1083 (1985). M. Shiobara, Agric. Biol. Chem., 16) F. K. Chu, R. B. Trimble and F. Maley, /. Biol. Chem., 253, 8691 (1978). 17) F. F. C. Wangand C. H. W. Hirs, J. Biol. Chem., 252, 8358 (1977).