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The glycoprotein nature of phospholipase A2, hyaluronidase and acid phosphatase from honey-bee venom
0041-0101/83 ß .00+ .00 0 1983 PerBamon P~eas Ltd .
Taxirnn, Vd . 21, No. 6, pp. 893-896, 1983. p,9nted ln p,nt griuin .
THE GLYCOPROTEIN NATURE OF PHOSPHOLIPASE A2, HYALURONIDASE AND ACID PHOSPHATASE FROM HONEYBEE VENOM LEOPOLD MÄRZ,' CHRISTIAN KÜHNE Snd HERIBERT MICHL Institut für Chemie, Universität für Bodenkultur, Gregor-M~ndelstrasse 33, A-1180 Vienna, Austria (Accepted for publication 26 Apri! 1983) L . M.vtz, C . Külwe and H . MICHL . The glycoprotein nature of phospholipase A hyaluronidase and acid phosphatase from honey-bee venom . Toxicon 21, 893-896, 1983 . - Experiments with immobilized concanavalin A strongly suggest a glycoprotein nature of three honey-bee venom enzymes, phospholipase A,, hyaluronidase'and acid phosphatase. The electrophoretitxlly and chromatographically detectable heterogeneity of phospholipase A, results from absence of carbohydrate in a subfraction . Mannose, fucose and N-acetylglucosamine, but not galactose nor N-acetylgalactosamine, are prescht in the con A-binding fraction of bee venom . It is therefore concluded that only N-glycosidically linked carbohydrate oa~urs in bee venom glycoproteins .
PREVIOUS reports suggested the presence of covalently bound carbohydrate in the protein components of the honey-bee (Apis mell~ca), with the evidence strongest in the case of phospholipase A~ (SHIPOLINI et al., 1974). There are indications, but no direct evidence, for glycocomponents in other bee venom enzymes (KRYSTEVA et al., 1973 ; SHKENDEROV et al., 1979). This communication presents data establishing the glycoprotein nature of phospholipase A~, hyaluronidase and acid phosphatase. Furthermore, it is demonstrated that carbohydrate is absent in a fraction of the phospholipase . Pooled lyophilized whole venom (batch 00380) was purchased from H . Mack (Illertissen, German Federal Republic) . Its protein content was 96 .4%, determined according to the method of LOWRY et al. (1951) . Neutral sugars (2.9%) were found in the dialyzed venom, assayed by the phenol - sulfuric acid method (Dusols et al., 1956). The activity of hyaluronidase (EC 3.2.1.35) (64.5 units per mg of protein) was determined turbidimetrically (TOLKSDORF et al., 1949; KASS and $EASTONE, 1944). One enzyme unit reduces the turbidity-causing capacity of 0.2 mg hyaluronic acid to that of 0.1 mg in 30 min at 37°C . Phospholipase activity (EC 3.1.1 .4) (179 .5 units per mg of protein) was calculated on the basis of the amount of fatty acid liberated from egg yolk lecithin (Sigma, Munich, GFR) and titrated in a pH-stat (GROSSMAN et al., 1974). One unit reflects the consumption of one micromole of NaOH per min . Acid phosphatase activity (EC 3 .1 .3.2 .) (0.54 units per mg of protein) was checked by measuring the cleavage of p-nitrophenyl phosphate (LINHART SIId WALTER, 1962; AMES, 19C~ at pH 4.8, the optimum of the enzyme . One unit liberates 1 Eunole of p-nitrophenol per min at 37°C. Slab gel electrophoresis on polyacrylamide (PAGE) was run at pH 4.3 (REISFELD et al., 1962) or on gels containing ~I'o whom all correspondence regarding this paper should be directed . 893
Short Communications
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3z
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S-Con A-I
r S-Con A-2
FIG. 1 . CHROMATOGRAPHY OF HONEY-BEE VENOM (0 .96 mg) ON SEPHAROSE 4B~ONCANAVALIN A. Column volume : 1 .9 ml; eluant: 0.02 M Tris -HCl/0 .5 M NaCI, pH 7.4 . The arrow indicates the addition of 0.5 M a-methyhnannoside to the eluant. Flow rate: 2 drops per min; fraction volume : 1 .2 ml . ~ ~ Acid phosphatase; f " hyaluronidase; 0 O phospholipase A, . Units are activity units.
sodium dodecyl sulfate (SDS) (LAENIIvILI, 1970). Proteins were stained with Amidoschwartz lOB or Coomassie Brilliant Blue R-250 (Bioltad, Vienna, Austria) and glycocomponents with periodate-Schiff reagents (FAIRBANKS et al., 1971). Enzymes were located on the pH 4.3-gels by extraction followed by activity assay. The monosaccharides were analyzed by gas-liquid chromatography as the corresponding alditol acetates (LEHNHARDT and WINZLER, 1968). On a column of Sepharose 4B~oncanavalin A (S-con A) (Pharmacia, Sweden), bee venom yields two fractions (Fig . 1). One peak appears unretarded (Son A-1), which contains melittin, apamin, other peptides and 38% of the phospholipase activity . No other enzyme was found in this fraction . By elution with a-methylmannoside, all of the hyaluronidase and acid phosphatase and the rest of the phospholipase activity (62%) were recovered (Scon A-2) . Identical results were obtained with various amounts of venom, provided the column capacity of 3.8 mg of venom per ml of gel was not exceeded . Both Soon A fractions were further chromatographed on Sephadex G-75 (Pharmacia) and compared with a sample of whole venom run under identical conditions (Fig . 2). On polyacrylamide gel, all enzyme fractions stain carbohydrate-positive except for the phospholipase A,-portion, which passes through S-con A without retardation (Fig. 1, S-con A-1). On PAGE and gel chromatography it co-migrates with the phospholipsse activity in peak F IIIb of the G-7S chromatogram . Its molecular weight, determined by SDS-PAGE, is 1S,S00, compared with 18,500 for the carbohydrate-positive phospholipase in Soon A-2, which in turn corresponds with F IIIa . No neutral or amino sugars were detected in S-con A-1, whereas the retarded peak (S-con A-2) contained mannose, fucose and N-acetylglucosamine in a ratio of 3 .0 : 0.52 : 2.05. These results demonstrate a glycoprotein nature for the three enzymes investigated, with the exception of one phospholipase subfraction. Its lack of con A-binding can be explained
Short Communications
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I .2
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0 .6 0.4
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FIO . 2. CHROMATOGRAPHY OF HONEY-ßEE VENOM (3O mg) ON SEPHADF.X G-7S . Column : 1.5 x 95 cm; chiant: 0.1 M ammonium formiate buffer, pH 4.6 . Flow rate : 8.0 ml/hr. Fraction volume in the void volume : 4.4 ml . O O Absorbante at 278 nm . For other symbols see legend to FtG. 1. FIV and FV represent melittin and apamin, respectively.
by either total absence of sugar or the masking of con A-bindiltg determinants due to differences in monosaccharide linkages . Negative carbohydrate analysis, negative periodate - Schiff reaction and the distinctly lower molecular weight lead us to believe that this fraction is devoid of carbohydrate. We were unable to discover any differences in enzyme specificity between the two phospholipase species. The presence of mannose and N-acetylglucosamine and the absence of Nacetylgalactosamine and galactose in the carbohydrate strongly suggest that it is of the asparagine-linked `N-glycan' type (MONTREUIL, 1980) only. Experiments are in progress to isolate and characterize the carbohydrate moieties of bee venom glycoproteins. Acknowledgements - This work was supported by grant no. 4613 of the Fonds zurFörderung der österreichischen Forschung, Vienna, Austria.
REFERENCES AMFS, B. N. (1966) Assay of inorganic phosphate, total phosphate and phosphatases . In : Methods In Enrymology, Vol. VIII, p. 115 (Cowwtctc, S. P. and KAPLAN, N. O., Eds.) . New York: Academic Press. Duaots, M., GILLES, K. A., HAMn.TON, J. K., REHERS, P. A. and SMITH, F. (1956) Colorimetric method for determination of sugars and related substances . Arratyt . Chem . 2i, 330. FAIRBANSS, G., STECac, T. and WALLACH, D. F. H. (1971) Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane. Biochemistry 10, 2606 . GROSSINANN, S., OFST'REICHEA, H. and SnVGER, T. P. (1974) Determination of the activity of phospholipases A, C, and D. Meth . biochtm. Analysis 22, 177.
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Short Communications
Kwss, E. H. and SEwsroxE, C. V. (1944) The role of the muooid polysaccharide (hyaluronic acid) in the virulence of group A hemolytic Streptococci . J. exp. Med. 79, 319. Kxvs~rEVw, M. A., MESeoa, B. K., Ivwlvov, C. P. and Sx~IVnEROV, S. (1973) Partial characterization of hyaluronidase from bee venom. C.r. Acad. bulg. Sci. 26, 917. LwEn~tnn.I, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680. LExtvxwRnr, W. F. 8IId WWZLER, R. J. (1968) DetcrminAt~ on of neutral sugars in glycoproteins by gas-liquid chromatography . J. Chromat. 34, 471 . LIIV~IwIeoT, K. and WwcrEx, K. (1982) Phosphatasen . In : Methoden der en~matischex Analyse, p. 779 (BEaontEVEa, H. U., Ed .) . Weinheim, GFR: Verhlg Chemie . Lowßv, O. H., RasEaaouox, N. J., FwxR, A. L. and RANDALL, R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265. MONrREUIL, J. (1980) Primary structure of glycoprotein glycans. Adv. Carbohyd. Chem . Biochem. 37, 157. REISFELD, R. A., LEwIS, U. J. and WILLIAMS, D. E. (1962) Disc electrophoresis of basic proteins and peptides on polyacrylamide gels . Nature 195, 281. SHIPOLWI, R. A., CALLEWAERT, G. L., C07TRELL, R. C. and VERNON, C. A. (1974) The amino acid sequence and carbohydrate content of phospholipase A, from bee venom. Eur. J. Biochem. 4E, 465. SxKENDEROV, S., IVANOVA, I. 8IId GRYGOROVA, K. (1979) An acid monophosphoesterase and o-glucosidase enzymes newly isolated from bee venom. Toxieon 17 (Suppl . 1), 169. ToLxsooteF, S., MCCREADY, M. H., McCuLLwax, D. R. and SCHWENK, E. (1949) The turbidimetric assay of hyaluronidase J. Lab. clip. Med. 34, 74 .
Taxirnn, Vd . 21, No. 6, pp. 893-896, 1983. p,9nted ln p,nt griuin .
THE GLYCOPROTEIN NATURE OF PHOSPHOLIPASE A2, HYALURONIDASE AND ACID PHOSPHATASE FROM HONEYBEE VENOM LEOPOLD MÄRZ,' CHRISTIAN KÜHNE Snd HERIBERT MICHL Institut für Chemie, Universität für Bodenkultur, Gregor-M~ndelstrasse 33, A-1180 Vienna, Austria (Accepted for publication 26 Apri! 1983) L . M.vtz, C . Külwe and H . MICHL . The glycoprotein nature of phospholipase A hyaluronidase and acid phosphatase from honey-bee venom . Toxicon 21, 893-896, 1983 . - Experiments with immobilized concanavalin A strongly suggest a glycoprotein nature of three honey-bee venom enzymes, phospholipase A,, hyaluronidase'and acid phosphatase. The electrophoretitxlly and chromatographically detectable heterogeneity of phospholipase A, results from absence of carbohydrate in a subfraction . Mannose, fucose and N-acetylglucosamine, but not galactose nor N-acetylgalactosamine, are prescht in the con A-binding fraction of bee venom . It is therefore concluded that only N-glycosidically linked carbohydrate oa~urs in bee venom glycoproteins .
PREVIOUS reports suggested the presence of covalently bound carbohydrate in the protein components of the honey-bee (Apis mell~ca), with the evidence strongest in the case of phospholipase A~ (SHIPOLINI et al., 1974). There are indications, but no direct evidence, for glycocomponents in other bee venom enzymes (KRYSTEVA et al., 1973 ; SHKENDEROV et al., 1979). This communication presents data establishing the glycoprotein nature of phospholipase A~, hyaluronidase and acid phosphatase. Furthermore, it is demonstrated that carbohydrate is absent in a fraction of the phospholipase . Pooled lyophilized whole venom (batch 00380) was purchased from H . Mack (Illertissen, German Federal Republic) . Its protein content was 96 .4%, determined according to the method of LOWRY et al. (1951) . Neutral sugars (2.9%) were found in the dialyzed venom, assayed by the phenol - sulfuric acid method (Dusols et al., 1956). The activity of hyaluronidase (EC 3.2.1.35) (64.5 units per mg of protein) was determined turbidimetrically (TOLKSDORF et al., 1949; KASS and $EASTONE, 1944). One enzyme unit reduces the turbidity-causing capacity of 0.2 mg hyaluronic acid to that of 0.1 mg in 30 min at 37°C . Phospholipase activity (EC 3.1.1 .4) (179 .5 units per mg of protein) was calculated on the basis of the amount of fatty acid liberated from egg yolk lecithin (Sigma, Munich, GFR) and titrated in a pH-stat (GROSSMAN et al., 1974). One unit reflects the consumption of one micromole of NaOH per min . Acid phosphatase activity (EC 3 .1 .3.2 .) (0.54 units per mg of protein) was checked by measuring the cleavage of p-nitrophenyl phosphate (LINHART SIId WALTER, 1962; AMES, 19C~ at pH 4.8, the optimum of the enzyme . One unit liberates 1 Eunole of p-nitrophenol per min at 37°C. Slab gel electrophoresis on polyacrylamide (PAGE) was run at pH 4.3 (REISFELD et al., 1962) or on gels containing ~I'o whom all correspondence regarding this paper should be directed . 893
Short Communications
894
0 .3
Is
3z
12
24
16
4
8
S-Con A-I
r S-Con A-2
FIG. 1 . CHROMATOGRAPHY OF HONEY-BEE VENOM (0 .96 mg) ON SEPHAROSE 4B~ONCANAVALIN A. Column volume : 1 .9 ml; eluant: 0.02 M Tris -HCl/0 .5 M NaCI, pH 7.4 . The arrow indicates the addition of 0.5 M a-methyhnannoside to the eluant. Flow rate: 2 drops per min; fraction volume : 1 .2 ml . ~ ~ Acid phosphatase; f " hyaluronidase; 0 O phospholipase A, . Units are activity units.
sodium dodecyl sulfate (SDS) (LAENIIvILI, 1970). Proteins were stained with Amidoschwartz lOB or Coomassie Brilliant Blue R-250 (Bioltad, Vienna, Austria) and glycocomponents with periodate-Schiff reagents (FAIRBANKS et al., 1971). Enzymes were located on the pH 4.3-gels by extraction followed by activity assay. The monosaccharides were analyzed by gas-liquid chromatography as the corresponding alditol acetates (LEHNHARDT and WINZLER, 1968). On a column of Sepharose 4B~oncanavalin A (S-con A) (Pharmacia, Sweden), bee venom yields two fractions (Fig . 1). One peak appears unretarded (Son A-1), which contains melittin, apamin, other peptides and 38% of the phospholipase activity . No other enzyme was found in this fraction . By elution with a-methylmannoside, all of the hyaluronidase and acid phosphatase and the rest of the phospholipase activity (62%) were recovered (Scon A-2) . Identical results were obtained with various amounts of venom, provided the column capacity of 3.8 mg of venom per ml of gel was not exceeded . Both Soon A fractions were further chromatographed on Sephadex G-75 (Pharmacia) and compared with a sample of whole venom run under identical conditions (Fig . 2). On polyacrylamide gel, all enzyme fractions stain carbohydrate-positive except for the phospholipase A,-portion, which passes through S-con A without retardation (Fig. 1, S-con A-1). On PAGE and gel chromatography it co-migrates with the phospholipsse activity in peak F IIIb of the G-7S chromatogram . Its molecular weight, determined by SDS-PAGE, is 1S,S00, compared with 18,500 for the carbohydrate-positive phospholipase in Soon A-2, which in turn corresponds with F IIIa . No neutral or amino sugars were detected in S-con A-1, whereas the retarded peak (S-con A-2) contained mannose, fucose and N-acetylglucosamine in a ratio of 3 .0 : 0.52 : 2.05. These results demonstrate a glycoprotein nature for the three enzymes investigated, with the exception of one phospholipase subfraction. Its lack of con A-binding can be explained
Short Communications
895
I .4
I .2
60 50
08
40
0 .6 0.4
20
0.2
10
FIO . 2. CHROMATOGRAPHY OF HONEY-ßEE VENOM (3O mg) ON SEPHADF.X G-7S . Column : 1.5 x 95 cm; chiant: 0.1 M ammonium formiate buffer, pH 4.6 . Flow rate : 8.0 ml/hr. Fraction volume in the void volume : 4.4 ml . O O Absorbante at 278 nm . For other symbols see legend to FtG. 1. FIV and FV represent melittin and apamin, respectively.
by either total absence of sugar or the masking of con A-bindiltg determinants due to differences in monosaccharide linkages . Negative carbohydrate analysis, negative periodate - Schiff reaction and the distinctly lower molecular weight lead us to believe that this fraction is devoid of carbohydrate. We were unable to discover any differences in enzyme specificity between the two phospholipase species. The presence of mannose and N-acetylglucosamine and the absence of Nacetylgalactosamine and galactose in the carbohydrate strongly suggest that it is of the asparagine-linked `N-glycan' type (MONTREUIL, 1980) only. Experiments are in progress to isolate and characterize the carbohydrate moieties of bee venom glycoproteins. Acknowledgements - This work was supported by grant no. 4613 of the Fonds zurFörderung der österreichischen Forschung, Vienna, Austria.
REFERENCES AMFS, B. N. (1966) Assay of inorganic phosphate, total phosphate and phosphatases . In : Methods In Enrymology, Vol. VIII, p. 115 (Cowwtctc, S. P. and KAPLAN, N. O., Eds.) . New York: Academic Press. Duaots, M., GILLES, K. A., HAMn.TON, J. K., REHERS, P. A. and SMITH, F. (1956) Colorimetric method for determination of sugars and related substances . Arratyt . Chem . 2i, 330. FAIRBANSS, G., STECac, T. and WALLACH, D. F. H. (1971) Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane. Biochemistry 10, 2606 . GROSSINANN, S., OFST'REICHEA, H. and SnVGER, T. P. (1974) Determination of the activity of phospholipases A, C, and D. Meth . biochtm. Analysis 22, 177.
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Short Communications
Kwss, E. H. and SEwsroxE, C. V. (1944) The role of the muooid polysaccharide (hyaluronic acid) in the virulence of group A hemolytic Streptococci . J. exp. Med. 79, 319. Kxvs~rEVw, M. A., MESeoa, B. K., Ivwlvov, C. P. and Sx~IVnEROV, S. (1973) Partial characterization of hyaluronidase from bee venom. C.r. Acad. bulg. Sci. 26, 917. LwEn~tnn.I, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680. LExtvxwRnr, W. F. 8IId WWZLER, R. J. (1968) DetcrminAt~ on of neutral sugars in glycoproteins by gas-liquid chromatography . J. Chromat. 34, 471 . LIIV~IwIeoT, K. and WwcrEx, K. (1982) Phosphatasen . In : Methoden der en~matischex Analyse, p. 779 (BEaontEVEa, H. U., Ed .) . Weinheim, GFR: Verhlg Chemie . Lowßv, O. H., RasEaaouox, N. J., FwxR, A. L. and RANDALL, R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265. MONrREUIL, J. (1980) Primary structure of glycoprotein glycans. Adv. Carbohyd. Chem . Biochem. 37, 157. REISFELD, R. A., LEwIS, U. J. and WILLIAMS, D. E. (1962) Disc electrophoresis of basic proteins and peptides on polyacrylamide gels . Nature 195, 281. SHIPOLWI, R. A., CALLEWAERT, G. L., C07TRELL, R. C. and VERNON, C. A. (1974) The amino acid sequence and carbohydrate content of phospholipase A, from bee venom. Eur. J. Biochem. 4E, 465. SxKENDEROV, S., IVANOVA, I. 8IId GRYGOROVA, K. (1979) An acid monophosphoesterase and o-glucosidase enzymes newly isolated from bee venom. Toxieon 17 (Suppl . 1), 169. ToLxsooteF, S., MCCREADY, M. H., McCuLLwax, D. R. and SCHWENK, E. (1949) The turbidimetric assay of hyaluronidase J. Lab. clip. Med. 34, 74 .