Eur. J. Riochem. 205, 729-735 (1992) (C) FEBS 1992
Purification and characterization of membrane-bound endoglycoceramidase from Corynebacterium sp. Hisashi ASHIDA ’, Kenji YAMAMOTO’, Hidehiko KUMAGAI’ and Tatsurokuro TOCHIKURA’ Rescarch Laboratory of Higashimaru Shoyu Co. Ltd, Hyogo, Japan Department of Food Science and Technology, Faculty of Agriculture, Kyoto University, Japan (Received November 18, 1991/January 22, 1992) - EJB 91 3559
Endoglycoceramidase catalyzes the hydrolysis of the linkage between oligosaccharides and ceramides of various glycosphingolipids. We found that a bacterial strain Corynebucterium sp., isolated from soil, produced endoglycoceramidase both intracellularly and extracellularly . The intracellular enzyme bound to the cell membrane was solubilized with 1YOTriton X-100 and purified to homogeneity about 170-fold with 60% recovery. The molecular mass of the enzyme was approximately 65 kDa. The enzyme is most active at pH 5.5-6.5 and stable at pH 3.5-8.0. Various neutral and acidic glycosphingolipids were hydrolyzed by the enzyme in the presence of 0.1% Triton X-100. Ganglioand lacto-type glycosphingolipids were readily hydrolyzed, but globo-type glycosphingolipids were hydrolyzed slowly.
Glycosphingolipids are widely found in the cell surface membranes of mammalian tissues and organs, and are especially abundant in neural tissues. They are amphipathic compounds consisting of oligosaccharide and ceramide moieties. Glycosphingolipids may play some roles in antigenic determinants concerned with cancer and blood groups, and are specific receptors for bacterial toxins and viruses. Recent progress in glycolipid research has revealed that glycosphingolipids may function as biological modulators for cell proliferation, recognition, adhesion and differentiation [I -71. The glycosphingolipid-degrading enzyme, tentatively called endoglycoceramidase or ceramide glycanase, is a new endo-type glycosidase that catalyzes the hydrolysis of the linkage between oligosaccharides and ceramides of various glycosphingolipids. The enzyme is very important as an analytical tool for the elucidation of biological functions and structures of glycosphingolipids. Only a few sources of the enzyme, Rhodococcus sp. [8, 91, leeches [lo, 111 and earthworms [12], have been found so far. The only enzyme from a microbial origin is that of Rhodococcus sp. Although the enzyme had been purified from a mutant strain of the actinomycete, purification from culture fluid proved very complicated [9]. Purification from the parent
strain was previously unsuccessful [8] because the strain produces the enzyme only in the presence of an inducer such as bovine brain acetone powder that made the enzyme purification difficult owing to the formation of (g1yco)lipid-enzyme complexes. Moreover, the actinomycete secretes a large amount of hemolysin into the culture medium. Thus, we searched for a better source of the glycosphingolipid-degrading enzyme produced without addition of inducers in the culture medium and found that a bacterial strain, Corynebacterium sp. A809, isolated from soil, produced a high level of the enzyme. Moreover, the enzyme bound to the membranes was easily purified to homogeneity. We report here the localization of a glycosphingolipid-degrading enzyme, endoglycoceramidase, in Corynebacterium sp., its relatively simple purification and some properties.
MATERIALS AND METHODS Materials
A mixture of gangliosides was prepared from bovine brains by the method of Ledeen and Yu [13]. Lactosylceramide, glucosylceramide and ceramide were purchased from Sigma Chemical. The following glycosphingolipids were gifts: GMlr Correspondenw to H. Ashida, Research Laboratory of Higashimaru Shoyu Co., Ltd, Torninaga, Tatsuno, Tatsuno, Hyogo, GMZand GDlawere from Sumitomo Seika; asialo-GMl, GM3, Forssman hapten, globoside and sialosyl-puru-globoside were Japan 679-41 Ahhreviutions. GD1,, (IV 3NeuAc, 11 3NeuAcGgOse4Cer). Nfrom Dr Y. Hirabayashi, Shizuoka College of Pharmacy, acctylneuraminosylgalactosyl-N-acctylgalactosaminyl-(~-acetylneu- Japan. rdminosy1)galactosylghcosykeramide ;G M l , (I1 3NeuAcGgOse4Cer), DEAE-Cellulofine AH was purchased from Chisso. galactosyl - N - acetylgalactosaminyl - ( N - acety1neuraminosyl)galac- Sephadex G-200 and G-150 were from Pharmacia Fine tosylglucosylccramide; GMz.(TI 3NeuAcGgOse3Cer), N-acetylgalacChemicals. Triton X-100 was from Nacalai Tesque. BCAIM tOSaminyl-(~-aCetylneUrdminOSyl)gdlactOSylglUCOSylCeramide; GM3, protein assay reagent was from Pierce. TLC plates were from (I1 3NeuAcLacCer), N-acelylneuraminosylgalactosylglucosylcerMerck. AmpureTMDT, the detergent-removing column, was amide; FAB-MS, fast-atom-bombardment/mass spectrometry. Ennzq’rnes. Endoglycoceramidase, glycosylcerarnidase, glycosyl-N- from Amersham. All other chemicals were of the highest grade acylsphingosine glycohydrolase (EC 3.2.1.62). available from commercial sources.
730 Microorganism and cultivation
Analytical methods
A bacterial strain, A809, which was isolated from a soil sample, was used throughout this study. This bacterium was Gram-positive, and rod/coccus growth cycle was found during growth in media. Its cell wall peptidoglycan contained mesodiaminopimelic acid, and major cell wall sugars were arabinose and galactose. From these results it was taxonomically identified as Corynehacterium sp. by referring to Bergey's Manual of Systematic Bacteriology [14]. Detailed characteristics will be reported elsewhere. The bacterium was inoculated into a 20-ml test tube containing 5 ml of a medium consisting of 0.5% glucose, 0.5% peptone, 0.5% yeast extract, 0.2% meat extract and 0.2% NaCl (pH 6.5). The culture was carried out for 6 days at 28' C with shaking, then the broth was transferred to a 2-1 Sakaguchi flask containing 500 ml of the same medium. The cultivation was continued under the same conditions as described above.
For detection of the ceramide, the reaction mixture was analyzed by TLC plate (Merck 5553) using chloroform/methanol(95 :5, by vol.) as the developing solvent. Ceramides were stained with Coomassie brilliant blue R by the method of Nakamura and Handa [19]. The oligosaccharide released from GMlwas analyzed by positive fast-atom-bombardment mass spectrometry (FABMS) using a Jeol JMS HX-100 mass spectrometer (Jeol Ltd, Japan) [9]. Diethanolamine was used as the matrix.
Endoglycoceramidase assay
So lub iliza t ion of endogly co ceram iduse
Endoglycoceramidase activity was assayed according to the method of ito and Yamagata 191. GMIwas used as the substrate. The reaction mixture contained 40 nmol GMland an appropriate amount of the enzyme in 60 pI 10 mM sodium acetate buffer (pH 6.5) containing 0.1% (mass/vol.) Triton X-100. After incubation at 37°C for 15 min, the reaction was terminated by the addition of 200 p1 carbonate/cyanide (pH 11.O), and the reducing power produced in the reaction mixture was measured by the method of Park-Johnson [15]. For the control mixture, the substrate solution was incubated for the indicated time, then the alkaline solution was added, followed by the enzyme preparation. The amount of oligosaccharides released from the glycosphingolipids is expressed as the increase in reducing power because the enzyme preparation was completely free from various exoglycosidases. 1 U enzyme activity was defined as the amount of enzyme which catalyzes the release of 1 pmol reducing power (as glucose)/min from GM,under the conditions described above. To determine substrate specificity, a reaction mixture containing 40 nmol various glycosphingolipids and 0.75 mU of the enzyme in 60 p1 10 mM sodium acetate buffer (pH 6.5) including 0.2% (mass/vol.) Triton X-100 was incubated at 37 C for 30 min and 48 h, and 10-pl aliquots were analyzed for digestion products by TLC using chloroform/methanol/ 0.2% CaC1, (4:4: 1, by vol.) as the developing solvent. Glycosphingolipids and oligosaccharides were revealed by spraying TLC plate with orcinol/H2S0, and scanning them with a Shimadzu CS-9000 chromatoscanner with the reflectance mode set at 540 nm. The degradation was calculated as follows: degradation ( O h ) = A , x 100/Al A 2 , where A1 is peak area of oligosaccharide released and A 2 is peak area of remaining substrate.
60 g wet cell paste of Corynebucterium sp. A809, which was harvested by centrifugation from 5 1 culture broth, was suspended in 10 mM potassium phosphate buffer containing 1.0% (mass/vol.) Triton X-100. The cells were shattered by sonic oscillation for 10 min. After gentle stirring for 2 h, the cell lysate was centrifuged at 24000 x g for 40 min to remove the cell debris. The supernatant solution was used as a crude enzyme preparation.
+
Other enzyme assays Other exoglycosidase activities were assayed using various p-nitrophenyl glycosides as substrates [16]. Protease activity was determined using milk casein as the substrate [17].
Purification of endoglycoceramidase Unless otherwise indicated, 10 mM potassium phosphate buffer (pH 7.0) containing 0.1 % (mass/vol.) Triton X-100 (buffer A) was used and all purification steps were performed at about 4°C.
DEAE-Cellul?fne A H column chromutogruphj
The enzyme solution was applied to a column (2.2 cm x 20 cm) of DEAE-Cellulofine AH previously equilibrated with buffer A. The column was washed with the same buffer followed by elution with a step-wise gradient of 0.1 0.3 M NaCl in buffer A. The major enzyme was eluted with 0.2 M NaCl in buffer A. The active fractions were pooled and concentrated to 30 ml by ultrafiltration with a YM-10 membrane (Amicon). Sephadex G-200 ge1,filtration The concentrated enzyme was applied to a column (2.0 cm x 110 cm) of Sephadex (3-200, previously equilibrated with buffer A then eluted with the same buffer. All fractions containing enzyme activity were pooled and dialyzed against 1 mM potassium phosphate buffer (pH 7.0) containing 0.1 YO (mass/vol.) Triton X-100. Hydrox-yaputite column chromutogruphy The dialyzed enzyme solution was applied to a hydroxyapatite column (1.2 cm x 7 cm) previously equilibrated with 1 mM potassium phosphate buffer (pH 7.0) containing 0.1% (mass/vol.) Triton X-100. The column was washed with the same buffer then eluted with a linear concentration gradient of 1 -1000 mM phosphate buffer. The major enzyme was eluted with about 15 mM phosphate buffer. Active fractions were concentrated and dialyzed against buffer A. The dialyzed enzyme was used as the purified enzyme.
Measurement of protein PAGE The protein concentration was measured using BCATM Pol yacrylamide disc-gel electrophoresis was performed by protein assay reagent with bovine serum albumin as the stanthe method of Davis [20]in 7.5% polyacrylamide with Tris,' dard [18].
73 1
Cultivation Time (days) Fig. 1. Time course of endoglycoceramidase production and cell growth of Corynebacteviurn sp. The culture was carried out in a 500-ml flask containing 100 ml medium at 28°C on a reciprocal shaker. Intracellular endoglycoceramidase activity was measured using solubilized enzyme prepared as described under Materials and Methods. Extracellular endoglycoceramidase activity was measured using culture fluid which was previously dialyzed against buffer A. (0)pH; ( A ) cell growth; ( 0 )intracellular endoglycoceramidasc; (0)extracellular cndoglycoceramidase.
Fig. 2. Localization of endoglycoceramidase in Corynebucterium sp. cells. Fraction 1 : cell debris after centrifugation (1800 x g, 5 min) with cell homogenate. Fraction 2: precipitate after ultracentrifugation (68000 x g, 60 min) with the supernatant. Fraction 3, supernatant after ultracentrifugation. Each fractions was assayed for endoglycoceramidase activity by TLC using chloroform/methanol/0.2% CaCI2 (4: 4: 1, by vol.). Glycosphingolipids and oligosaccharides were revealed by spraying the TLC plate with orcinol/H2SO4 reagent. Lane 1, G M l ;lane 2, GMland purified enzyme; lane 3, GMl and fraction 1; lane 4, fraction 1; lane 5, GMl and fraction 2; lane 6, fraction 2; lane 7, GMl and fraction 3 ; lane 8, fraction 3.
glycine buffer (pH 8.3). SDS/polyacrylamide slab-gel electrophoresis was performed in 12.5% acrylamide and 0.1 % SDS with a discontinuous Tris/glycine buffer system by the method of Laemmli [21]. Gels were stained for protein with Coomassie brilliant blue R. Molecular mass determination
The molecular mass of the enzyme was determined using gel-permeation HPLC on a TSK G3000SWxL column (0.78 cm x 30 cm) equilibrated with I0 mM potassium phosphate buffer (pH 7.0) containing 0.1% Triton X-100 and 0.1 M NaCl by the method of Andrews [22]. The column was calibrated with ferritin (450 kDa), catalase (250 kDa), aldolase (158 kDa) and bovine serum albumin (67 kDa). The molecular mass of the enzyme was also estimated by SDS/PAGE. The following standards were used: phosphorylase /> (94 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa), soybean trypsin inhibitor (20.1 kDa) and dactoalbumin (14.4 kDa).
RESULTS Production of endogl ycoceramidase
The time course of endoglycoceramidase production and cell growth of Corynehacterium sp. were investigated under standard culture conditions. As shown in Fig. 1, endoglycoceramidase activity was found in both culture broth and cells. The intracellular enzyme activity increased with cell growth and attained a maximum after 6 days. The extracellular enzyme also increased with cell growth, though it was produced at a level of about six times less than that of the intracellular enzyme.
Fig. 3. TLC of ceramide released from GMl by endoglycoceramidase. The enzyme reaction was carried out at 37°C for 6 h in the presence of 0.1 YOTriton X-100. Ceramide released from GMl by the enzyme was analyzed by TLC using chloroform/methanol (95: 5 , by vol.) as the developing solvent. Ceramides were stained with Coomassie brilliant blue R. Lane I, G M l ; lane 2, authentic ceramide (from bovine brain); lane 3, GMl and enzyme; lane 4, enzyme.
conditions at 28°C for 6 days. Cells were washed twice and suspended in phosphate-buffered saline (pH 7.0), followed by sonic disruption at 0°C for 10 min. The cell homogenate was centrifuged at 1800 x g for 5 min and cell debris (fraction I) was obtained. The resulting supernatant was centrifuged once more at 68000 x g for 60 min to obtain the membrane (fraction 2) and supernatant (fraction 3). The cell debris and membrane were suspended in potassium phosphate buffer (pH 7.0) and analyzed. Each fraction was incubated with substrate GMl and analyzed for endoglycoceramidase activity by TLC (Fig. 2 ) . Fractions 1 and 2 had high activity levels, whereas fraction 3 had none. These results indicate that the intracellular endoglycoceramidase of the bacterium is the membranebound enzyme.
Localization of endoglycoceramidase in Coryncbactwium sp. cells
Identification of reaction products
Localization of endoglycoceramidase in Corynebacterium sp. cells was investigated using cells grown under aerobic
The ceramide and oligosaccharide released from G, by the enzyme were identified. After the enzyme reaction, the
132
Fig.4. FAB-MS of oligosaccharide released from Cu, by endoglycoceramidase. The mass ion at m/r 1021 and m/z 1104 are from (M + Na)' and (M + DEAH)*. The molecular mass of 998 Da calculated from these values coincided with the thcorctical value of G M lohgosaccharide. M.G M loligosaccharide; DEAH, diethanolamine + hydrogcn.
Table 1 . Purification of endoglycoceramidase.
Step
Protein
Activity
Specific activity
Purification
Yield
mg
mU
mU/mg
-fold
Yo
Solubilized enzyme 1780 DEAE-Cellulofine 12.4 AH Scphadex (3-200 7.95 H ydroxyapatite 6.25
3550
2990 2316 2110
1.99
1
100
41.30
20.8 146 170
84
291.3 337.6
65 59
reaction mixture was evaporated to dryness and dissolved in 10 pl chloroform/methanol (2: I , by vol.). The solution was analyzed for ceramide by TLC using chloroform/methanol (95: 5, by vol.) as the developing solvent. The TLC plate was dried and stained with Coomassie brilliant blue R (Fig. 3). Only one spot released from GMlwas observed and its position corresponded to authentic ceramide from bovine brain. The oligosaccharide derived from GMlby enzymatic hydrolysis was purified by gel filtration on Sephadex G-25 and analyzed by FAB-MS (Fig. 4). Its molecular mass was found to be 998 Da, calculated from a m/z 1021 for [Fig. 4, ( M + Na)'] and mi. 1104 for [Fig. 4, (M DEAH)'] in H 2 0 ,which coincided completely with the theoretical value of GMloligosaccharide. These findings indicate that the enzyme from Corynebncterium sp. produces intact ceramides and oligosaccharides by the cleavage of glycosphingolipids.
Fig. 5. Native PAGE (A) and SDS/PAGE (B) of purified endoglycoceramidase. (A) Purified enzyme (10 pg) was applied to a 7.5"h polyacrylamide gel. (B) Purified enzyme (10 keg) was subjected to SDS/ PAGE with a 12.5% polya,crylamide gel. The protein standards (STD) used were phosphorylase h (molecular mass 94 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30kDa), soybean trypsin inhibitor (20.1 kDa) and rc-lactoalbumin (14.4kDa).
+
Purification of endoglycoceramidase
lntracellular endoglycoceramidase was purified using the cell homogenate supernatant as the starting preparation. A purification summary of the endoglycoceramidase is shown in Table 1. The enzyme was purified about 170-fold from thc solubilizcd fraction.
Enzyme purity and molecular mass
The purified enzyme migrated as an almost single protein band on polyacrylamide disc-gel electrophoresis, as shown in Fig. 5A. No exoglycosidasc or protease activities were observed in the final preparation. SDS/PAGE of the enzyme also gave a single protein band corresponding to a molecular mass of about 65 kDa (Fig. 5R). When the HPLC on a TSK G3000SWx,, column was used for molecular mass determination, the enzyme was eluted between ferritin (450 kDa) and catalase (250 kDa). These results
733 Table 2. Effects of detergents on endoglycoceramidase activity. The cn7yme preparation was previously treated with AmpurerM DT to rcniove Triton X-100. Enzymc activity was assaycd in thc presence of 0.2% (mass/vol.) detergents using GMl as thc substrate at 37°C for 15 min.
De t ergeii t
Relativc activity
Yo None Sodium cholalc Sodium deoxycholaic Sodium taurocholate Sodium taurodeoxycholate Uriji-58 Twccn 20 Nonidet P-40 Triton X-100
10.7 15.1 10.6 11.5
14.7 7.8 21.8 97.4 100
suggest that the enzyme consists of some identical polypeptides or aggrcgates. Properties of the enzyme Maximal activity was obtained at pH 5.5-6.5 in 10 niM sodium acetate buffer including 0.2% (mass/vol.) Triton X-100 using GM1as the substrate. The enzyme was stable in the pH range 4.5- 8.0 when maintained at 37°C for 30 min in various buffers (10 mM) contained 0.2% (mass/vol.) Triton X-1 00. The thermal stability of the enzyme was examined after incubating a t various temperatures for 10 min (pH 7.0). The cnzyme was stable up to about 45°C and completely inactive above 60 “C. Full activity was retained after repeated freezing/ thawing and the enzyme was stored frozen at - 20°C for over a year without any loss of activity. The effects of metal ions on endoglycoceramidase showed that Hg’.’ and Zn” (1 mM) inhibited 85% and 50% of the activity. respectively. The other metal ions (1 mM) tested (Mg”, C a ” , Pb”, CoZt,M n Z c , Cu”, F e z ’ , CdZt and Cr3 ’) had little or no effect on the enzyme activity. The effect of substrate concentration on the rate of hydrolysis was examined using various concentrations of G M 1 . The K , was calculated to be 0.15 m M from a LineweaverBurk plot. Effects of various detergents The effects of various detergents on the enzyme activity were examined using GM1as the substrate and the enzyme preparation from which Triton X-100 was removed with AmpureTMDT. Among various detergents tested a t a concentration of 0.2% (massivol.), Triton X-100 was the most effective and Nonidet P-40 which has a very similar structure to that of Triton X-100 also enhanced the enzyme activity. Briji58, Tween 20 and bile salts at the same concentration were ineffective (Table 2). The enzyme had abou22z* of the activity when Triton X-100 was removed. When thc effect of various Triton X-100 concentrations on enzyme activity was cxamined, the enzyme was most active at a concentration of 0.20/0. Substrate specificity To investigate the substrate specificity, purified enzyme wits incubated with various neutral and acidic glyco-
Fig. 6. Degradation of various glycosphingolipids by endoglycoceramidase. Various glycosphingolipids were incubated with purified enzyme at 37°C for 6 h. After incubation, 10 p1 reaction mixture was analyzed for the digestion products by TLC using chloroforni/ methanol/0.2% CaClz (4:4: 1, by vol.). Glycosphingolipids and oligosaccharides were visualizcd by spraying the TLC plate with orcinol/H2S04 reagent. Lane 1, Gola; lane 2, G D l rand enzyme; lane 3, G M l ;lane 4, GMl and cnzyme; lane 5 , GY2;lane 6, GM2and cnzyme; lane 7, G u 3 ; lane 8, GM3and enzymc, lane 9 ; asialo-GM1; lane 10, asialo-GMl and enzyme; lane 11, sialosyl-pura-globoside; lane 12, sialosyl-para-globoside and cnzyme; lane 13, globoside; lane 14, globosidc and enzyme; lane 15, Forssman hapten; lane 16, Forssman hapten and cnzyme.
sphingolipids in the presence of 0.2% Triton X-100 (mass/ vol.), then analyzed by TLC as described in Materials and Methods (Fig. 6). Only one oligosaccharide was produced from each substrate by hydrolysis of this enzyme. The enzyme acted on both neutral and acidic glycosphingolipids. Table 3 summarizes extent of the hydrolysis of various glycosphingolipids by the enzyme. The enzyme readily hydrolyzed ganglio-type glycosphingolipids except G M 3 , which was hydrolyzed at a slower rate than others. Sialosyl-purugloboside was also readily degraded by the enzyme. Globotype glycosphingolipids were slowly hydrolyzed. Similarly, the enzyme cleaved lactosylceramide slowly, but failed to degrade glucosylceramide.
DISCUSSION Endoglycoceramidase was first found in the culture fluid of Rhodococcus sp. [8] and was purified from its mutant strain [9]. A similar enzyme which was called ceramide glycanase, was also found in leeches [lo, 111 and earthworms [12]. Although Rhodococcus sp. endoglyeoceramidase was the sole enzyme of microbial origin, this strain required gangliosides as the inducer to produce the enzyme [8].Adding gangliosides to microorganism culture media is impractical since gangliosides are difficult to obtain. Moreover, the purification of the enzyme from the culture fluid of the strain was reportedly unsuccessful owing to the formation of (g1yco)lipid-enzyme complex during the purification procedure [8]. To solve this problem, Ito and Yamagata obtained a mutant of Rhodococcus sp. that produced endoglycoceramidase in the absence of inducers, after which they purified the enzyme. However, this procedure was rather complex because it required nine steps [9]. We searched for microorganisms producing endoglycoceramidase constitutively and found a bacterial strain, Corynebacterium sp. A809, from soil. This strain produced only low levels of exogenous enzymc. However, high levels o f the
734 Table 3. Substrate specificity of endoglycoceramidase. Glycosphingolipids (40 nmol each) were incubated with 0.75 mU enzyme in 60 pI 10 m M sodium acetate buffer (pH 6.5) including 0.2% (mass/vol.) Triton X-100 at 37°C. After 30 min (condition 1) and 48 h (condition 2) incubation, 10 p1 aliquots were analyzed by TLC followed by scanning with a chromatoscanner. The percentage degradation was calculated as described in Materials and Methods. Substrate
Substrate
Structure
Hydrolysis extent after incubation for
tYPC
Ganglio
~
GD1,
G Mt GMZ GM3
asialo-GMl Lacto Globo
sialosyl-paragloboside
Forssman hapten globoside
_
_
_
30 min
48 h
NeuActu2+3Galbl -t3GalNAc~l+4(NeuAca2+3)Gal~1+4Glc~l+ lCer GalPl+3GalNAclI +4(NeuAca2+3)Gal~1+4GIc~l+lCer GalNAc~l+4(NeuAca2+3)Gal~l +4Glcp1+ 1Cer NeuAc~2+3Gal/?1+4Glc~l+lCer Gall1 +3GalNAc~1+4Gal~1+4GlcfiI+ICer
69.7 83.0 75.1 1.5 88.9
100 100 100 84.9
NcuAca2+3Gal~1+4GlcNAc~1-t3Galj?1+4Glc~1+1 Cer
78.5
100
GalNAcil+3GalNAc~I+3Galal+4Gal~1+4GlcBl + I Ccr GalNAcPl+3Galal +4Galb1+4Glc/ll+ lCer
0.9
ceramide trihexoside hctosykeramide glucos ylceramide
enzyme were found intracellularly (2.4 Ujl, which is six times higher than that in culture fluid 0.4 Ujl). Though the intracellular enzyme was tightly bound to the cell membrane, it was easily solubilized by sonication in the presence of 1.O% Triton X-100. After solubilization, it was purified to homogencity with high recovery by a relatively simple procedure. The purified enzyme required detergents for expression of the full activity as do other glycosphingolipid-degrading enzymes. Triton X-100 was the most effective detergent for enhancing the enzyme activity. When Triton X-100 was removed from the enzyme solution, the solution became turbid within a short time. Therefore, this detergent may play roles in the solubility of both substrates and enzyme molecules which seemed to be rather hydrophobic. Recently, specific activator proteins which stimulate Rhodococcus sp. endoglycoceramidase activity in the absence of detergents have been found in the culture fluid of the actinomycete [23]. On the other hand, when Corynebacterium sp. cells carrying endoglycoceramidase were allowed to act on GMM1 without Triton X-100, the substrate was degraded at nearly the same rate as in the presence of the detergent. This suggested that the Coqwehactcrium cell itself may have some activators similar to detergents. The substrate specificity of the Corynebacterium sp. enzyme was very similar to that of the Rhodococcus sp. enzyme. The endoglycoceramidase of Corynebacterium sp. hydrolyzes the glycoceramide linkage of ganglio-, globo- and lacto-type glycosphingolipids to produce both intact oligosaccharides and ceramides. Although the enzyme slowly hydrolyzed lactosylceramide, glucosylceramide was not hydrolyzed at all. Thc Fact indicates that the enzyme is different f r o m
glycosylceramidase, which hydrolyzes the linkage between monosaccharide and ceramide. Both enzymes of Rhodococcus sp. and leech also could not hydrolyze glucosylceramide 19, 111. Corynebacteriurn sp. enzyme seemed readily to hydrolyze glycosphingolipids having long sugar chains (above four residues), although the globo-type was hydrolyzed slowly. This phenomenon was found in the enzymes of both
Gala1 +4Gal~l+4Glc/Jl+ICer G a l p l + 4 G l c ~ 1--t 1Cer GlcBl+ 1Cer
1.4
1.8 1 .0
0
_
100
75.8 38.6 79.9 54.3 0
Rhodococcus sp. and leech. The b-linked N-acetylgalactosamine neighboring ceramidetrihexoside may reduce the access of the enzyme to the substrate. Although glycosphingolipids are full of variety, it may simplify the analysis of oligosaccharides and ceramides after the cleavage of glycospingolipids by this enzyme. This could then lead to the elucidation of the roles of endogenous glycosphingolipids in living cell surfaces using this enzyme. We thank Dr M. Ito, Mitsubishi Kasei Institute of Life Sciencc. for his hclp in carrying out FAB-MS.
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735 15. Park, J. T. & Johnson, M. J. (1949) J. Biol. Chem. 181, 249-
151. 16. Li, Y.-T. & Li, S.-C. (1972) M e t h o d Enzymol. 28, 702-113. 17. Knunitz, M . (1947) J . Gen. Physiol. 30, 291 -310. 18. Smith, P. K., Krohnb, R . I., Hermanson, G. T., Mallia, A. K., Gartncr, F. H.. Provenxano, M. D., Fujimoto, E. K., Goeke, N. M., Olson, B. J. & Klenk, D. C. (1985) Anal. Biochem. 150, 76 - 85.
19. Nakamura, K . & Handa, S. (1984) Anal. Biochem. 142, 406410. 20. Davis, B. J . (1964) Ann. N . Y . Acad. Sci. 121, 404-407. 21. Laemmli, U. K. (1970) Nature 227,680-685. 22. Andrews, p. (1964) Biochem. J . 91,222-223. 23. rto, M., Jkegami, Y. & Yamagata, T. (1991) J . Biol. Chem. 266, 7919-7926.
Purification and characterization of membrane-bound endoglycoceramidase from Corynebacterium sp. Hisashi ASHIDA ’, Kenji YAMAMOTO’, Hidehiko KUMAGAI’ and Tatsurokuro TOCHIKURA’ Rescarch Laboratory of Higashimaru Shoyu Co. Ltd, Hyogo, Japan Department of Food Science and Technology, Faculty of Agriculture, Kyoto University, Japan (Received November 18, 1991/January 22, 1992) - EJB 91 3559
Endoglycoceramidase catalyzes the hydrolysis of the linkage between oligosaccharides and ceramides of various glycosphingolipids. We found that a bacterial strain Corynebucterium sp., isolated from soil, produced endoglycoceramidase both intracellularly and extracellularly . The intracellular enzyme bound to the cell membrane was solubilized with 1YOTriton X-100 and purified to homogeneity about 170-fold with 60% recovery. The molecular mass of the enzyme was approximately 65 kDa. The enzyme is most active at pH 5.5-6.5 and stable at pH 3.5-8.0. Various neutral and acidic glycosphingolipids were hydrolyzed by the enzyme in the presence of 0.1% Triton X-100. Ganglioand lacto-type glycosphingolipids were readily hydrolyzed, but globo-type glycosphingolipids were hydrolyzed slowly.
Glycosphingolipids are widely found in the cell surface membranes of mammalian tissues and organs, and are especially abundant in neural tissues. They are amphipathic compounds consisting of oligosaccharide and ceramide moieties. Glycosphingolipids may play some roles in antigenic determinants concerned with cancer and blood groups, and are specific receptors for bacterial toxins and viruses. Recent progress in glycolipid research has revealed that glycosphingolipids may function as biological modulators for cell proliferation, recognition, adhesion and differentiation [I -71. The glycosphingolipid-degrading enzyme, tentatively called endoglycoceramidase or ceramide glycanase, is a new endo-type glycosidase that catalyzes the hydrolysis of the linkage between oligosaccharides and ceramides of various glycosphingolipids. The enzyme is very important as an analytical tool for the elucidation of biological functions and structures of glycosphingolipids. Only a few sources of the enzyme, Rhodococcus sp. [8, 91, leeches [lo, 111 and earthworms [12], have been found so far. The only enzyme from a microbial origin is that of Rhodococcus sp. Although the enzyme had been purified from a mutant strain of the actinomycete, purification from culture fluid proved very complicated [9]. Purification from the parent
strain was previously unsuccessful [8] because the strain produces the enzyme only in the presence of an inducer such as bovine brain acetone powder that made the enzyme purification difficult owing to the formation of (g1yco)lipid-enzyme complexes. Moreover, the actinomycete secretes a large amount of hemolysin into the culture medium. Thus, we searched for a better source of the glycosphingolipid-degrading enzyme produced without addition of inducers in the culture medium and found that a bacterial strain, Corynebacterium sp. A809, isolated from soil, produced a high level of the enzyme. Moreover, the enzyme bound to the membranes was easily purified to homogeneity. We report here the localization of a glycosphingolipid-degrading enzyme, endoglycoceramidase, in Corynebacterium sp., its relatively simple purification and some properties.
MATERIALS AND METHODS Materials
A mixture of gangliosides was prepared from bovine brains by the method of Ledeen and Yu [13]. Lactosylceramide, glucosylceramide and ceramide were purchased from Sigma Chemical. The following glycosphingolipids were gifts: GMlr Correspondenw to H. Ashida, Research Laboratory of Higashimaru Shoyu Co., Ltd, Torninaga, Tatsuno, Tatsuno, Hyogo, GMZand GDlawere from Sumitomo Seika; asialo-GMl, GM3, Forssman hapten, globoside and sialosyl-puru-globoside were Japan 679-41 Ahhreviutions. GD1,, (IV 3NeuAc, 11 3NeuAcGgOse4Cer). Nfrom Dr Y. Hirabayashi, Shizuoka College of Pharmacy, acctylneuraminosylgalactosyl-N-acctylgalactosaminyl-(~-acetylneu- Japan. rdminosy1)galactosylghcosykeramide ;G M l , (I1 3NeuAcGgOse4Cer), DEAE-Cellulofine AH was purchased from Chisso. galactosyl - N - acetylgalactosaminyl - ( N - acety1neuraminosyl)galac- Sephadex G-200 and G-150 were from Pharmacia Fine tosylglucosylccramide; GMz.(TI 3NeuAcGgOse3Cer), N-acetylgalacChemicals. Triton X-100 was from Nacalai Tesque. BCAIM tOSaminyl-(~-aCetylneUrdminOSyl)gdlactOSylglUCOSylCeramide; GM3, protein assay reagent was from Pierce. TLC plates were from (I1 3NeuAcLacCer), N-acelylneuraminosylgalactosylglucosylcerMerck. AmpureTMDT, the detergent-removing column, was amide; FAB-MS, fast-atom-bombardment/mass spectrometry. Ennzq’rnes. Endoglycoceramidase, glycosylcerarnidase, glycosyl-N- from Amersham. All other chemicals were of the highest grade acylsphingosine glycohydrolase (EC 3.2.1.62). available from commercial sources.
730 Microorganism and cultivation
Analytical methods
A bacterial strain, A809, which was isolated from a soil sample, was used throughout this study. This bacterium was Gram-positive, and rod/coccus growth cycle was found during growth in media. Its cell wall peptidoglycan contained mesodiaminopimelic acid, and major cell wall sugars were arabinose and galactose. From these results it was taxonomically identified as Corynehacterium sp. by referring to Bergey's Manual of Systematic Bacteriology [14]. Detailed characteristics will be reported elsewhere. The bacterium was inoculated into a 20-ml test tube containing 5 ml of a medium consisting of 0.5% glucose, 0.5% peptone, 0.5% yeast extract, 0.2% meat extract and 0.2% NaCl (pH 6.5). The culture was carried out for 6 days at 28' C with shaking, then the broth was transferred to a 2-1 Sakaguchi flask containing 500 ml of the same medium. The cultivation was continued under the same conditions as described above.
For detection of the ceramide, the reaction mixture was analyzed by TLC plate (Merck 5553) using chloroform/methanol(95 :5, by vol.) as the developing solvent. Ceramides were stained with Coomassie brilliant blue R by the method of Nakamura and Handa [19]. The oligosaccharide released from GMlwas analyzed by positive fast-atom-bombardment mass spectrometry (FABMS) using a Jeol JMS HX-100 mass spectrometer (Jeol Ltd, Japan) [9]. Diethanolamine was used as the matrix.
Endoglycoceramidase assay
So lub iliza t ion of endogly co ceram iduse
Endoglycoceramidase activity was assayed according to the method of ito and Yamagata 191. GMIwas used as the substrate. The reaction mixture contained 40 nmol GMland an appropriate amount of the enzyme in 60 pI 10 mM sodium acetate buffer (pH 6.5) containing 0.1% (mass/vol.) Triton X-100. After incubation at 37°C for 15 min, the reaction was terminated by the addition of 200 p1 carbonate/cyanide (pH 11.O), and the reducing power produced in the reaction mixture was measured by the method of Park-Johnson [15]. For the control mixture, the substrate solution was incubated for the indicated time, then the alkaline solution was added, followed by the enzyme preparation. The amount of oligosaccharides released from the glycosphingolipids is expressed as the increase in reducing power because the enzyme preparation was completely free from various exoglycosidases. 1 U enzyme activity was defined as the amount of enzyme which catalyzes the release of 1 pmol reducing power (as glucose)/min from GM,under the conditions described above. To determine substrate specificity, a reaction mixture containing 40 nmol various glycosphingolipids and 0.75 mU of the enzyme in 60 p1 10 mM sodium acetate buffer (pH 6.5) including 0.2% (mass/vol.) Triton X-100 was incubated at 37 C for 30 min and 48 h, and 10-pl aliquots were analyzed for digestion products by TLC using chloroform/methanol/ 0.2% CaC1, (4:4: 1, by vol.) as the developing solvent. Glycosphingolipids and oligosaccharides were revealed by spraying TLC plate with orcinol/H2S0, and scanning them with a Shimadzu CS-9000 chromatoscanner with the reflectance mode set at 540 nm. The degradation was calculated as follows: degradation ( O h ) = A , x 100/Al A 2 , where A1 is peak area of oligosaccharide released and A 2 is peak area of remaining substrate.
60 g wet cell paste of Corynebucterium sp. A809, which was harvested by centrifugation from 5 1 culture broth, was suspended in 10 mM potassium phosphate buffer containing 1.0% (mass/vol.) Triton X-100. The cells were shattered by sonic oscillation for 10 min. After gentle stirring for 2 h, the cell lysate was centrifuged at 24000 x g for 40 min to remove the cell debris. The supernatant solution was used as a crude enzyme preparation.
+
Other enzyme assays Other exoglycosidase activities were assayed using various p-nitrophenyl glycosides as substrates [16]. Protease activity was determined using milk casein as the substrate [17].
Purification of endoglycoceramidase Unless otherwise indicated, 10 mM potassium phosphate buffer (pH 7.0) containing 0.1 % (mass/vol.) Triton X-100 (buffer A) was used and all purification steps were performed at about 4°C.
DEAE-Cellul?fne A H column chromutogruphj
The enzyme solution was applied to a column (2.2 cm x 20 cm) of DEAE-Cellulofine AH previously equilibrated with buffer A. The column was washed with the same buffer followed by elution with a step-wise gradient of 0.1 0.3 M NaCl in buffer A. The major enzyme was eluted with 0.2 M NaCl in buffer A. The active fractions were pooled and concentrated to 30 ml by ultrafiltration with a YM-10 membrane (Amicon). Sephadex G-200 ge1,filtration The concentrated enzyme was applied to a column (2.0 cm x 110 cm) of Sephadex (3-200, previously equilibrated with buffer A then eluted with the same buffer. All fractions containing enzyme activity were pooled and dialyzed against 1 mM potassium phosphate buffer (pH 7.0) containing 0.1 YO (mass/vol.) Triton X-100. Hydrox-yaputite column chromutogruphy The dialyzed enzyme solution was applied to a hydroxyapatite column (1.2 cm x 7 cm) previously equilibrated with 1 mM potassium phosphate buffer (pH 7.0) containing 0.1% (mass/vol.) Triton X-100. The column was washed with the same buffer then eluted with a linear concentration gradient of 1 -1000 mM phosphate buffer. The major enzyme was eluted with about 15 mM phosphate buffer. Active fractions were concentrated and dialyzed against buffer A. The dialyzed enzyme was used as the purified enzyme.
Measurement of protein PAGE The protein concentration was measured using BCATM Pol yacrylamide disc-gel electrophoresis was performed by protein assay reagent with bovine serum albumin as the stanthe method of Davis [20]in 7.5% polyacrylamide with Tris,' dard [18].
73 1
Cultivation Time (days) Fig. 1. Time course of endoglycoceramidase production and cell growth of Corynebacteviurn sp. The culture was carried out in a 500-ml flask containing 100 ml medium at 28°C on a reciprocal shaker. Intracellular endoglycoceramidase activity was measured using solubilized enzyme prepared as described under Materials and Methods. Extracellular endoglycoceramidase activity was measured using culture fluid which was previously dialyzed against buffer A. (0)pH; ( A ) cell growth; ( 0 )intracellular endoglycoceramidasc; (0)extracellular cndoglycoceramidase.
Fig. 2. Localization of endoglycoceramidase in Corynebucterium sp. cells. Fraction 1 : cell debris after centrifugation (1800 x g, 5 min) with cell homogenate. Fraction 2: precipitate after ultracentrifugation (68000 x g, 60 min) with the supernatant. Fraction 3, supernatant after ultracentrifugation. Each fractions was assayed for endoglycoceramidase activity by TLC using chloroform/methanol/0.2% CaCI2 (4: 4: 1, by vol.). Glycosphingolipids and oligosaccharides were revealed by spraying the TLC plate with orcinol/H2SO4 reagent. Lane 1, G M l ;lane 2, GMland purified enzyme; lane 3, GMl and fraction 1; lane 4, fraction 1; lane 5, GMl and fraction 2; lane 6, fraction 2; lane 7, GMl and fraction 3 ; lane 8, fraction 3.
glycine buffer (pH 8.3). SDS/polyacrylamide slab-gel electrophoresis was performed in 12.5% acrylamide and 0.1 % SDS with a discontinuous Tris/glycine buffer system by the method of Laemmli [21]. Gels were stained for protein with Coomassie brilliant blue R. Molecular mass determination
The molecular mass of the enzyme was determined using gel-permeation HPLC on a TSK G3000SWxL column (0.78 cm x 30 cm) equilibrated with I0 mM potassium phosphate buffer (pH 7.0) containing 0.1% Triton X-100 and 0.1 M NaCl by the method of Andrews [22]. The column was calibrated with ferritin (450 kDa), catalase (250 kDa), aldolase (158 kDa) and bovine serum albumin (67 kDa). The molecular mass of the enzyme was also estimated by SDS/PAGE. The following standards were used: phosphorylase /> (94 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa), soybean trypsin inhibitor (20.1 kDa) and dactoalbumin (14.4 kDa).
RESULTS Production of endogl ycoceramidase
The time course of endoglycoceramidase production and cell growth of Corynehacterium sp. were investigated under standard culture conditions. As shown in Fig. 1, endoglycoceramidase activity was found in both culture broth and cells. The intracellular enzyme activity increased with cell growth and attained a maximum after 6 days. The extracellular enzyme also increased with cell growth, though it was produced at a level of about six times less than that of the intracellular enzyme.
Fig. 3. TLC of ceramide released from GMl by endoglycoceramidase. The enzyme reaction was carried out at 37°C for 6 h in the presence of 0.1 YOTriton X-100. Ceramide released from GMl by the enzyme was analyzed by TLC using chloroform/methanol (95: 5 , by vol.) as the developing solvent. Ceramides were stained with Coomassie brilliant blue R. Lane I, G M l ; lane 2, authentic ceramide (from bovine brain); lane 3, GMl and enzyme; lane 4, enzyme.
conditions at 28°C for 6 days. Cells were washed twice and suspended in phosphate-buffered saline (pH 7.0), followed by sonic disruption at 0°C for 10 min. The cell homogenate was centrifuged at 1800 x g for 5 min and cell debris (fraction I) was obtained. The resulting supernatant was centrifuged once more at 68000 x g for 60 min to obtain the membrane (fraction 2) and supernatant (fraction 3). The cell debris and membrane were suspended in potassium phosphate buffer (pH 7.0) and analyzed. Each fraction was incubated with substrate GMl and analyzed for endoglycoceramidase activity by TLC (Fig. 2 ) . Fractions 1 and 2 had high activity levels, whereas fraction 3 had none. These results indicate that the intracellular endoglycoceramidase of the bacterium is the membranebound enzyme.
Localization of endoglycoceramidase in Coryncbactwium sp. cells
Identification of reaction products
Localization of endoglycoceramidase in Corynebacterium sp. cells was investigated using cells grown under aerobic
The ceramide and oligosaccharide released from G, by the enzyme were identified. After the enzyme reaction, the
132
Fig.4. FAB-MS of oligosaccharide released from Cu, by endoglycoceramidase. The mass ion at m/r 1021 and m/z 1104 are from (M + Na)' and (M + DEAH)*. The molecular mass of 998 Da calculated from these values coincided with the thcorctical value of G M lohgosaccharide. M.G M loligosaccharide; DEAH, diethanolamine + hydrogcn.
Table 1 . Purification of endoglycoceramidase.
Step
Protein
Activity
Specific activity
Purification
Yield
mg
mU
mU/mg
-fold
Yo
Solubilized enzyme 1780 DEAE-Cellulofine 12.4 AH Scphadex (3-200 7.95 H ydroxyapatite 6.25
3550
2990 2316 2110
1.99
1
100
41.30
20.8 146 170
84
291.3 337.6
65 59
reaction mixture was evaporated to dryness and dissolved in 10 pl chloroform/methanol (2: I , by vol.). The solution was analyzed for ceramide by TLC using chloroform/methanol (95: 5, by vol.) as the developing solvent. The TLC plate was dried and stained with Coomassie brilliant blue R (Fig. 3). Only one spot released from GMlwas observed and its position corresponded to authentic ceramide from bovine brain. The oligosaccharide derived from GMlby enzymatic hydrolysis was purified by gel filtration on Sephadex G-25 and analyzed by FAB-MS (Fig. 4). Its molecular mass was found to be 998 Da, calculated from a m/z 1021 for [Fig. 4, ( M + Na)'] and mi. 1104 for [Fig. 4, (M DEAH)'] in H 2 0 ,which coincided completely with the theoretical value of GMloligosaccharide. These findings indicate that the enzyme from Corynebncterium sp. produces intact ceramides and oligosaccharides by the cleavage of glycosphingolipids.
Fig. 5. Native PAGE (A) and SDS/PAGE (B) of purified endoglycoceramidase. (A) Purified enzyme (10 pg) was applied to a 7.5"h polyacrylamide gel. (B) Purified enzyme (10 keg) was subjected to SDS/ PAGE with a 12.5% polya,crylamide gel. The protein standards (STD) used were phosphorylase h (molecular mass 94 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30kDa), soybean trypsin inhibitor (20.1 kDa) and rc-lactoalbumin (14.4kDa).
+
Purification of endoglycoceramidase
lntracellular endoglycoceramidase was purified using the cell homogenate supernatant as the starting preparation. A purification summary of the endoglycoceramidase is shown in Table 1. The enzyme was purified about 170-fold from thc solubilizcd fraction.
Enzyme purity and molecular mass
The purified enzyme migrated as an almost single protein band on polyacrylamide disc-gel electrophoresis, as shown in Fig. 5A. No exoglycosidasc or protease activities were observed in the final preparation. SDS/PAGE of the enzyme also gave a single protein band corresponding to a molecular mass of about 65 kDa (Fig. 5R). When the HPLC on a TSK G3000SWx,, column was used for molecular mass determination, the enzyme was eluted between ferritin (450 kDa) and catalase (250 kDa). These results
733 Table 2. Effects of detergents on endoglycoceramidase activity. The cn7yme preparation was previously treated with AmpurerM DT to rcniove Triton X-100. Enzymc activity was assaycd in thc presence of 0.2% (mass/vol.) detergents using GMl as thc substrate at 37°C for 15 min.
De t ergeii t
Relativc activity
Yo None Sodium cholalc Sodium deoxycholaic Sodium taurocholate Sodium taurodeoxycholate Uriji-58 Twccn 20 Nonidet P-40 Triton X-100
10.7 15.1 10.6 11.5
14.7 7.8 21.8 97.4 100
suggest that the enzyme consists of some identical polypeptides or aggrcgates. Properties of the enzyme Maximal activity was obtained at pH 5.5-6.5 in 10 niM sodium acetate buffer including 0.2% (mass/vol.) Triton X-100 using GM1as the substrate. The enzyme was stable in the pH range 4.5- 8.0 when maintained at 37°C for 30 min in various buffers (10 mM) contained 0.2% (mass/vol.) Triton X-1 00. The thermal stability of the enzyme was examined after incubating a t various temperatures for 10 min (pH 7.0). The cnzyme was stable up to about 45°C and completely inactive above 60 “C. Full activity was retained after repeated freezing/ thawing and the enzyme was stored frozen at - 20°C for over a year without any loss of activity. The effects of metal ions on endoglycoceramidase showed that Hg’.’ and Zn” (1 mM) inhibited 85% and 50% of the activity. respectively. The other metal ions (1 mM) tested (Mg”, C a ” , Pb”, CoZt,M n Z c , Cu”, F e z ’ , CdZt and Cr3 ’) had little or no effect on the enzyme activity. The effect of substrate concentration on the rate of hydrolysis was examined using various concentrations of G M 1 . The K , was calculated to be 0.15 m M from a LineweaverBurk plot. Effects of various detergents The effects of various detergents on the enzyme activity were examined using GM1as the substrate and the enzyme preparation from which Triton X-100 was removed with AmpureTMDT. Among various detergents tested a t a concentration of 0.2% (massivol.), Triton X-100 was the most effective and Nonidet P-40 which has a very similar structure to that of Triton X-100 also enhanced the enzyme activity. Briji58, Tween 20 and bile salts at the same concentration were ineffective (Table 2). The enzyme had abou22z* of the activity when Triton X-100 was removed. When thc effect of various Triton X-100 concentrations on enzyme activity was cxamined, the enzyme was most active at a concentration of 0.20/0. Substrate specificity To investigate the substrate specificity, purified enzyme wits incubated with various neutral and acidic glyco-
Fig. 6. Degradation of various glycosphingolipids by endoglycoceramidase. Various glycosphingolipids were incubated with purified enzyme at 37°C for 6 h. After incubation, 10 p1 reaction mixture was analyzed for the digestion products by TLC using chloroforni/ methanol/0.2% CaClz (4:4: 1, by vol.). Glycosphingolipids and oligosaccharides were visualizcd by spraying the TLC plate with orcinol/H2S04 reagent. Lane 1, Gola; lane 2, G D l rand enzyme; lane 3, G M l ;lane 4, GMl and cnzyme; lane 5 , GY2;lane 6, GM2and cnzyme; lane 7, G u 3 ; lane 8, GM3and enzymc, lane 9 ; asialo-GM1; lane 10, asialo-GMl and enzyme; lane 11, sialosyl-pura-globoside; lane 12, sialosyl-para-globoside and cnzyme; lane 13, globoside; lane 14, globosidc and enzyme; lane 15, Forssman hapten; lane 16, Forssman hapten and cnzyme.
sphingolipids in the presence of 0.2% Triton X-100 (mass/ vol.), then analyzed by TLC as described in Materials and Methods (Fig. 6). Only one oligosaccharide was produced from each substrate by hydrolysis of this enzyme. The enzyme acted on both neutral and acidic glycosphingolipids. Table 3 summarizes extent of the hydrolysis of various glycosphingolipids by the enzyme. The enzyme readily hydrolyzed ganglio-type glycosphingolipids except G M 3 , which was hydrolyzed at a slower rate than others. Sialosyl-purugloboside was also readily degraded by the enzyme. Globotype glycosphingolipids were slowly hydrolyzed. Similarly, the enzyme cleaved lactosylceramide slowly, but failed to degrade glucosylceramide.
DISCUSSION Endoglycoceramidase was first found in the culture fluid of Rhodococcus sp. [8] and was purified from its mutant strain [9]. A similar enzyme which was called ceramide glycanase, was also found in leeches [lo, 111 and earthworms [12]. Although Rhodococcus sp. endoglyeoceramidase was the sole enzyme of microbial origin, this strain required gangliosides as the inducer to produce the enzyme [8].Adding gangliosides to microorganism culture media is impractical since gangliosides are difficult to obtain. Moreover, the purification of the enzyme from the culture fluid of the strain was reportedly unsuccessful owing to the formation of (g1yco)lipid-enzyme complex during the purification procedure [8]. To solve this problem, Ito and Yamagata obtained a mutant of Rhodococcus sp. that produced endoglycoceramidase in the absence of inducers, after which they purified the enzyme. However, this procedure was rather complex because it required nine steps [9]. We searched for microorganisms producing endoglycoceramidase constitutively and found a bacterial strain, Corynebacterium sp. A809, from soil. This strain produced only low levels of exogenous enzymc. However, high levels o f the
734 Table 3. Substrate specificity of endoglycoceramidase. Glycosphingolipids (40 nmol each) were incubated with 0.75 mU enzyme in 60 pI 10 m M sodium acetate buffer (pH 6.5) including 0.2% (mass/vol.) Triton X-100 at 37°C. After 30 min (condition 1) and 48 h (condition 2) incubation, 10 p1 aliquots were analyzed by TLC followed by scanning with a chromatoscanner. The percentage degradation was calculated as described in Materials and Methods. Substrate
Substrate
Structure
Hydrolysis extent after incubation for
tYPC
Ganglio
~
GD1,
G Mt GMZ GM3
asialo-GMl Lacto Globo
sialosyl-paragloboside
Forssman hapten globoside
_
_
_
30 min
48 h
NeuActu2+3Galbl -t3GalNAc~l+4(NeuAca2+3)Gal~1+4Glc~l+ lCer GalPl+3GalNAclI +4(NeuAca2+3)Gal~1+4GIc~l+lCer GalNAc~l+4(NeuAca2+3)Gal~l +4Glcp1+ 1Cer NeuAc~2+3Gal/?1+4Glc~l+lCer Gall1 +3GalNAc~1+4Gal~1+4GlcfiI+ICer
69.7 83.0 75.1 1.5 88.9
100 100 100 84.9
NcuAca2+3Gal~1+4GlcNAc~1-t3Galj?1+4Glc~1+1 Cer
78.5
100
GalNAcil+3GalNAc~I+3Galal+4Gal~1+4GlcBl + I Ccr GalNAcPl+3Galal +4Galb1+4Glc/ll+ lCer
0.9
ceramide trihexoside hctosykeramide glucos ylceramide
enzyme were found intracellularly (2.4 Ujl, which is six times higher than that in culture fluid 0.4 Ujl). Though the intracellular enzyme was tightly bound to the cell membrane, it was easily solubilized by sonication in the presence of 1.O% Triton X-100. After solubilization, it was purified to homogencity with high recovery by a relatively simple procedure. The purified enzyme required detergents for expression of the full activity as do other glycosphingolipid-degrading enzymes. Triton X-100 was the most effective detergent for enhancing the enzyme activity. When Triton X-100 was removed from the enzyme solution, the solution became turbid within a short time. Therefore, this detergent may play roles in the solubility of both substrates and enzyme molecules which seemed to be rather hydrophobic. Recently, specific activator proteins which stimulate Rhodococcus sp. endoglycoceramidase activity in the absence of detergents have been found in the culture fluid of the actinomycete [23]. On the other hand, when Corynebacterium sp. cells carrying endoglycoceramidase were allowed to act on GMM1 without Triton X-100, the substrate was degraded at nearly the same rate as in the presence of the detergent. This suggested that the Coqwehactcrium cell itself may have some activators similar to detergents. The substrate specificity of the Corynebacterium sp. enzyme was very similar to that of the Rhodococcus sp. enzyme. The endoglycoceramidase of Corynebacterium sp. hydrolyzes the glycoceramide linkage of ganglio-, globo- and lacto-type glycosphingolipids to produce both intact oligosaccharides and ceramides. Although the enzyme slowly hydrolyzed lactosylceramide, glucosylceramide was not hydrolyzed at all. Thc Fact indicates that the enzyme is different f r o m
glycosylceramidase, which hydrolyzes the linkage between monosaccharide and ceramide. Both enzymes of Rhodococcus sp. and leech also could not hydrolyze glucosylceramide 19, 111. Corynebacteriurn sp. enzyme seemed readily to hydrolyze glycosphingolipids having long sugar chains (above four residues), although the globo-type was hydrolyzed slowly. This phenomenon was found in the enzymes of both
Gala1 +4Gal~l+4Glc/Jl+ICer G a l p l + 4 G l c ~ 1--t 1Cer GlcBl+ 1Cer
1.4
1.8 1 .0
0
_
100
75.8 38.6 79.9 54.3 0
Rhodococcus sp. and leech. The b-linked N-acetylgalactosamine neighboring ceramidetrihexoside may reduce the access of the enzyme to the substrate. Although glycosphingolipids are full of variety, it may simplify the analysis of oligosaccharides and ceramides after the cleavage of glycospingolipids by this enzyme. This could then lead to the elucidation of the roles of endogenous glycosphingolipids in living cell surfaces using this enzyme. We thank Dr M. Ito, Mitsubishi Kasei Institute of Life Sciencc. for his hclp in carrying out FAB-MS.
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