A Novel Glycosphingolipid-degrading Enzyme Cleaves of the Linkage between the Oligosaccharide and Ceramide of Neutral and Acidic Glycosphingolipids*

A novel glycosphingolipid-degrading enzyme was found in the cultured supernatant of Rhodococcus sp. G-74-2. It was purified 34.7-fold from the supernatant with 32.2% recovery by ammonium sulfate precipitation followed by Sephadex G-100 chromatography. The enzyme was demonstrated capable of cleaving the linkage between the oligosaccharide and ceramide of various acidic and neutral glycosphingolipids, producing intact oligosaccharides and ceramides. However, it was noted to hardly make any attack on linkages between monosaccharides and ceramides (cerebrosides) or between oligosaccharides and diacylglycerol (gly-coglycerolipids). The enzyme preparation was completely free from various exoglycosidases and pro- teases. Furthermore, it was found to degrade neither N-linked nor 0-linked glycoproteins. This enzyme, which is tentatively called endoglycoceramidase, should greatly facilitate the study of glycosphingo- lipids. oligosaccharides the Endogiyc~erami- eluted at the void active dialyzed 2 sodium acetate buffer, 6.0, and lyophilized. (kl, v/v) as a matrix and applied to the target. The atom gun was operated at 6 kV, using xenon as the bombarding gas. For determination of the activity toward glycopep- tides, 250 pg of each were incubated with 0.2 milliunits of the enzyme in 100 pl of 0.05 M sodium acetate buffer, pH 6.0, at 37 "C for 48 h. Their degradation by the enzyme was examined by TLC, using a solvent system of 1-butyl alcohol/acetic acid/HpO (2:1:1, v/v). Exo- glycosidases and proteases activities were assayed with p-nitrophen-ylglycosides (9) and Azocoll (10) as substrates, respectively.

A novel glycosphingolipid-degrading enzyme was found in the cultured supernatant of Rhodococcus sp. G-74-2. It was purified 34.7-fold from the supernatant with 32.2% recovery by ammonium sulfate precipitation followed by Sephadex G-100 chromatography. The enzyme was demonstrated capable of cleaving the linkage between the oligosaccharide and ceramide of various acidic and neutral glycosphingolipids, producing intact oligosaccharides and ceramides. However, it was noted to hardly make any attack on linkages between monosaccharides and ceramides (cerebrosides) or between oligosaccharides and diacylglycerol (glycoglycerolipids). The enzyme preparation was completely free from various exoglycosidases and proteases. Furthermore, it was found to degrade neither N-linked nor 0-linked glycoproteins. This enzyme, which is tentatively called endoglycoceramidase, should greatly facilitate the study of glycosphingolipids.
Glycosphingolipids are characteristic constituents of the outer surface of animal cells and assumed essential to cellcell recognition, antigenicity, and cell growth regulation (1, 2). StiIl, no methods are presently available for the isolation of intact oligosaccharides and ceramides from glycosphingolipids at the same time so as to study the structure and functions of glycosphingolipids. The present paper describes a novel enzyme, endoglycoceramidase, by which the linkage between the oligosaccharide and ceramide of acidic and neutral glycosphingolipids is cleaved. Use of this enzyme should greatly facilitate the study of glycosphingo~ipid functions and structure.
T. Hori, Shiga University, Japan; Glcal-4Glc~l-1Diacylglycerol, from Dr. T. Ogawa, Institute of Physical and Chemical Research, Japan. Enzyme Assay-Endoglycoceramidase activity was assayed using a mixture of bovine brain gangliosides as the substrate. The reaction mixture contained 150 pg of gangliosides, 50 Ng of sodium taurodeoxycholate, and an appropriate amount of the enzyme in 100 ~1 of 0.05 M sodium acetate buffer, pH 6.0. Following incubation at 37 "C for 1 h, the reducing power produced in an aliquot of the reaction mixture was measured by the method of Park and Johnson (7). One unit of the enzyme was defined as that quantity which would catalyze the release of 1 #mol of reducing power (as glucose) per min from gangliosides under the conditions described above. Since the enzyme preparation was completely free from various exoglycosidases, the amounts of glycosphingolipid-derived oligosaccharides were expressed in terms of the reducing power released from the glycosphingolipids.
.~ __ oligosaccharides from gangliosides by the enzyme. Endogiyc~eramidase was eluted at the void volume. The active fractions were pooled, dialyzed against 2 mM sodium acetate buffer, pH 6.0, and lyophilized. Since the enzyme activity was not solubilized in 0.1 M acetate buffer, pH 6.0, after lyophilization, the enzyme was extracted with octyl glucose from the lyophilized powder. A portion of the lyophilized powder (200 mg) was dissolved in 8 ml of 0.1 M acetate buffer, pH 6.0, containing 160 mg of octyl glucose and kept on ice for 3 h. The mixture was subjected to ultracentrifugation (183,500 X g for 30 min). The clear supernatant obtained was dialyzed against 5 m M acetate buffer, pH 6.0, and used for the present study.
Analytical Methods-The following solvent systems were used for TLC and HPTLC: 1-butyl alcohol/acetic acid/water (2:1:1, v/v) for oligosaccharides and chloroform/methanol (955, v/v) for ceramides from glycosphingolipids. Glycosphingolipids and oligosaccharides were made visible by spraying the TLC plates with orcinol-HzS04 reagent ( Fig. 2 A ) or diphenylamine reagent (Fig. 2B). Ceramides were stained with Coomassie Brilliant Blue by the method of Nakamura and Handa (8). The ceramides released from these glycosphingolipids were analyzed by negative fast atom bombardment-mass spectrometry, using a JEOL JMS HX-100 mass spectrometer (JEOL Ltd., Japan). The lower phase of the enzyme digestion products following partition with chloroform/methanol (23, v/v) was dried under a stream of nitrogen and dissolved in chloroform/methanoi (21, v/v) containing triethanolamine (kl, v/v) as a matrix and applied to the target. The atom gun was operated at 6 kV, using xenon as the bombarding gas. For determination of the activity toward glycopeptides, 250 pg of each were incubated with 0.2 milliunits of the enzyme in 100 pl of 0.05 M sodium acetate buffer, pH 6.0, at 37 "C for 48 h. Their degradation by the enzyme was examined by TLC, using a solvent system of 1-butyl alcohol/acetic acid/HpO (2:1:1, v/v). Exoglycosidases and proteases activities were assayed with p-nitrophenylglycosides (9) and Azocoll (10) as substrates, respectively.

RESULTS AND DISCUSSION
The enzyme was purified 34.7-fold from a culture filtrate of newly isolated soil actinomycetes of Rhodococcus sp. G-74-2 with 32.2% recovery by ammonium sulfate precipitation followed by Sephadex G-100 chromatography. The partially purified enzyme preparation was completely free from the following exoglycosidase activities: a-and @-galactosidases, a-and @-glucosidases, 0-N-acetylhexosaminidase, a-N-acetylgalactosaminidase, a-N-acetylglucosaminidase, a-L-fucosidase, and a-and ~-mannosidase. The enzyme was also free from protease activity. The general properties of the enzyme are as follows: optimal activity a t about pH 6.0 and stable between p H 5.0 and 9.0; potently inhibited by Hg", Zn2+, and Cu2+ (1 mM), but not so by Ba", M$+, Ca2+, and EDTA, all at the same concentration. The addition of taurodeoxycholate at the concentration of 0.5 mg/ml increased the enzyme activity about 2.5 times in comparison with that in the absence of the detergent when a mixture of bovine brain gangliosides was used as a substrate. Fig. 1 shows the time course for the degradation by this enzyme of asialo-GMl, G M~, and neogalatriaosylceramide. The degradation rate of glycosphingolipids was monitored by the reducing power produced from each glycosphingolipid through the action of the enzyme, since it degraded the glycosphingolipid to produce one oligosaccharide corresponding to an intact sugar chain of each glycosphingolipid, as will be discussed in detail later. The time courses of the degradation rates of asialo-GM, and GM, by the enzyme were quite similar. 91.5 and 94.9% of the sugar chains of these glycosphingolipids, respectively, were released, following incubation for 21 h under the conditions described in the legend to Fig. 1. From these results, it is apparent that the enzyme degrades both neutral and acidic glycosphingolipids to the same extent. It was also found to cleave the galactosylceramide linkage of neoga~atriaosylceramide, although the rate at which it cleaved the galactosylceramide linkage was somewhat slower (75.0% of the sugar chains were released, Fig. 1) than that of the Glycosphingolipids, 1 pmol each, were separately incubated at 37 "C with 4.2 milliunits of the enzyme in 1 ml of 0.05 M sodium acetate buffer, pH 6.0, containing 0.5 mg of taurodeoxycholate except in the case of the degradation of cerebrosides. For cerebrosides, 2.0 mg of taurodeoxycholate were present in the 1.0 ml of reaction mixture. At the times indicated, an aliquot of each reaction mixture was withdrawn for measurement of reducing power by the Park-Johnson method (7). Degradation percentage was calculated as follows: reducing power released (pmol of glucose) from the glycosphingolipid oligosaccharide content (pmol) x 100 in the glycosphingolipi~ -In this experiment, the molecular weights of asialo-GM,, GM,, neogalatriaosylceramide, and cerebroside were regarded as 1282,1573,1023, and 755, respectively. glucosylceramide linkages of other glycosphingolipids. Hardly any attack was made by the enzyme on the cerebrosides in which monosaccharides are linked to ceramides (Fig. 1). The amounts of ceramides released from these glycosphingolipids by the enzyme were also examined. The reaction mixture of asialo-GMl, GMl, and neog~atriaosylceramide with the enzyme following incubation for 21 h was applied onto a TLC plate which was then developed with a solvent containing chloroform/methanol (955, v/v). The TLC plate was dried and stained with Coomassie Brilliant Blue (8). Spots corresponding to authentic ceramides were scanned with a Shimadzu CS-930 chromatoscanner using a reflectance mode at 580 nm and quantified with known amounts of internal standards. As a result, 87.0,89.6, and 67.0% of the ceramides were found to be released from asialo-GM,, G M~, a n d neogalatriaosylceramides, respectively, through the action of this enzyme. The molar ratio of oligosaccharide (calculated from reducing power as described above) to ceramide released from each glycosphingolipid by digestion with the enzyme for 21 h was estimated to be 1:l.
The sugar compositions of the oligosaccharides released from aSidO-GMl, GMlr and neogalatriaosylceramide were analyzed before and after treatment with KBH, (Table I). The molar ratio of galactose, hexosamine, and glucose was about 2:l:l for oligosaccharides from aSialO-GMi and GM, before reduction, and 96.1 and 92.4% of the glucose of these oligosaccharides, respectively, were destroyed on reduction with KBHs. The galactose and hexosamine remained intact. The only constituent of oligosaccharide obtained from neogalatriaosylceramide was galactose, 34% of which was destroyed by reduction. This result demonstrates that the enzyme pro-TABLE I Analysis of oligosaccharides released from glycosphingolipids by endoglycoceramidase Glycosphingolipids, each in the amount of 100 nmol, were separately incubated with 0.42 milliunits of the enzyme in 100 pl of 0.05 M sodium acetate buffer, pH 6.0, containing taurodeoxycholate (0.5 mg/ml) at 37 "C for 21 h. Following incubation, each reaction mixture was partitioned with 5 volumes of chloroform/methanol (2:1, v/ v). The resulting upper layer was dried under a stream of nitrogen and subjected to sugar analysis before and after reduction with KBH,. Sugar component analysis was performed with an Altex high performance liquid chromatography system using an anion-exchange column following hydrolysis of oligosaccharides with 2.5 N trifluoroacetic acid at 100 "C for 14 h under nitroeen eas as Dreviouslv reDorted (16).
The data presented above clearly indicate that the enzyme cleaves the glucosylceramide linkage between the oligosaccharide and ceramide of acidic and neutral glycosp~~ngol~pids, producing both intact sugar chains and ceramides. it is of interest that the enzyme also cleaved the gafactosylceramide linkage of neogalatriaosylceramide, although at a slower rate. The activities of both these enzymes were recently found separable by Sephadex G-100 chromatography followed by DEAE-Sepharose chromatography (the details for this procedure will be reported elsewhere).
Enzymes that hydrolyze the linkage between monosaccharides and ceramides are called glycosylceramidase (EC 3.2.1.62) but they are incapable of hydrolyzing the linkage between oligosaccharides and ceramides in any glycosphingolipid (14, 15). However, the enzyme in the present research is more specific to the linkage between an oligosaccharide and ceramide than that between a monosaccharide and ceramide (Fig. 1) and thus we have tentatively designated it as an "endo-type glycosylceramidase," i.e. "endoglycosylceramidase" or "endoglycoceramidase" for short. Presently, it remains to be clarified whether cleavage activity toward the oligosaccharide-ceramide and monosaccharide-ceramide linkages as observed in the present research (Table I) is the action of two instead of only one enzyme. Further purification of the endoglycoceramidase i s in progress. Nevertheless, the enzyme we have discovered should greatly assist the study of glycosphingolipids.