Endo-type glycosylceramidases (EC 3.2.1.123) are a unique class of enzymes that specifically cleave the glycosidic linkage between oligosaccharide and ceramide in glycosphingolipids (GSLs). The enzymes present in Rhodococcus sp. (Ito and Yamagata, 1986) and Corynebacterium sp. (Ashida et al., 1992) have been designated as endoglycoceramidase (EGCase) and those in leech (Li et al., 1986) and earthworm (Li et al., 1987), as ceramide glycanase (CGase). They have been found useful for the structural analysis of GSLs (Shimamura et al., 1988; Fukaya et al., 1989; Higashi et al., 1990) and elucidation of biological functions of cell surface GSLs (Ponce et al., 1993; Muramoto et al., 1994; Ji et al., 1995). The glycan substrate specificity of EGCases and CGases has been done (Ito and Yamagata, 1986; Li et al., 1986, 1987; Ito and Yamagata, 1989; Ashida et al., 1992), but the importance of hydrophobic structure for these enzymes has yet to be fully determined owing to heterogeneity in the ceramide structure and the absence of glycolipid analogs. The enzymes also catalyze the transglycosylation of oligosaccharides to alcohol, a useful step in the preparation of neoglycoconjugates to test the biological functions of GSL oligosaccharides (Li et al., 1991; Ashida et al., 1993). However, lipophilic alcohols have been used occasionally as acceptors, and thus the substrate specificity towards the aglycone remains to be characterized in detail. In this regard, well-designed artificial GSL analogs may provide information on hydrophobic aglycone structures as substrates for EGCases/CGase. Novel amphipathic lactosides were recently developed with the N-acyl chain introduced at the position corresponding to that of lactosylceramide (LacCer) and mimicked GSL (Miura et al., 1996). Artificial glycolipids, such as n-octyl lactoside, undergo degradation by endo-type glycosylceramidases (Zhou et al., 1989; Carter et al., 1992). To determine more precisely the substrate specificity of EGCase/CGase, synthetic neoglycolipids were tested as substitutes. The use of suitable lipophilic substitutes as substrates for EGCase/CGase may facilitate affinity ligand construction and the preparation of neoglycolipids through transglycosylation by these enzymes. In this study, artificial amphipathic lactosides with different aglycone structures were tested with endo-type glycosylceramidases from Rhodococcus sp., Corynebacterium sp., and leech. The substrate specificity of EGCases from microorganisms toward aglycone structures differed from that of CGase from leech. The chromogenic lactoside, 2-N-hexadecanoylamino-4-nitrophenyl-O-[beta]-lactoside, was hydrolyzed by both EGCases and CGase and thus may be useful for detecting the activity of endo-type glycosylceramidases.
Amphipathic lactosides
LacCer is the simplest GSL structure that is susceptible to EGCases and CGase. It has been shown that a single alkyl chain may be the necessary aglycone for CGase (Li and Li, 1994). Although both enzymes are specific for glycolipids, the contribution of the ceramide portion to substrate recognition remains unclear. The importance of the acylamide structure in ceramides on the rates of EGCase/CGase-catalyzed hydrolysis was tested using N-acyl-aminoethyl lactosides (CnamEtLac), N-acyl-aminoethyl thiolactosides (CnamEt-S-Lac), and N-acyl-aminobutyl lactosides (CnamBuLac ), where n shows the length of the acyl chain. The first two compounds each possesses an N-acylamide bond at the same site as in the intact ceramide, whereas in the third, the distance between the amide and glycosidic oxygen increases from C2 to C4. Using CnamBuLac as a substrate for these enzymes should thus provide some indication of the significance of the acylamide structure and its position in ceramides. n-Alkyl lactosides (CnLac) each possessing a lactosyl moiety and alkyl chains of different length (even carbon number of C4-C18 ) were used as synthetic substrates for comparison of hydrolysis rates, since they have been found to be capable of assessing the substrate specificity of endo-type glycosylceramidases. Figure
Figure 1. Structures of lactosylceramide and synthetic lactosides. GCase- or CGase-catalyzed hydrolysis of synthetic amphipathic lactosides
Synthetic amphipathic lactosides were examined for susceptibility to EGCases from Rhodococcus sp. and Corynebacterium sp. and to CGase from leech under conditions optimal for the hydrolysis of LacCer, as specified in previous studies (Li et al., 1986; Ito and Yamagata, 1989; Ashida et al., 1992). As shown in Figure
Figure 2. Enzymatic hydrolysis of synthetic lactosides. Synthetic [beta]-lactosides (50 nmol) were incubated with the enzymes at 37°C for 16 h. The extent of hydrolysis was determined as described in Materials and methods. Left panel (A and C), hydrolysis of n-alkyl lactosides (CnLac); right panel (B and D), hydrolysis of N-acylaminoethyl [beta]-lactoside (CnamEtLac), C16amEt-S-Lac (S16), C8amBuLac (8Bu), C12amBuLac (12Bu). Open bars, EGCase from Rhodococcus sp., 3 mU; solid bars, EGCase from Corynebacterium sp., 1 mU, in (A) and (B); solid bars, CGase from leech, 0.3 mU in (C) and (D). The numbers on the abscissa indicate the length of chain, n, of the lactosides.
Both microbial EGCases showed essentially the same capacity to hydrolyze lactosides, whereas this capacity for CGase from leech was determined by the particular aglycone structure. CGase hydrolyzed n-alkyl and N-acylaminoethyl lactosides at basically the same rate (Figure
Table I.
Enzyme from
Km
Reported Km for GM1
Leech
28 µM
15 µM
Zhou et al., 1989
Corynebacterium sp.
1.4 mM
0.15 mM
Ashida et al., 1992
Rhodococcus sp.
2.9 mM
0.5 mMa
Ito and Yamagata, 1989
Chromogenic substrate for endo-type glycosylceramidases
p-Nitrophenyl lactoside is resistant to EGCase- and CGase-catalyzed hydrolysis and thus there is no commercially available chromogenic substrate for EGCase/CGase. Considering the above findings, the p-nitrophenyl lactoside derivative, 2-N-hexadecanoylamino-4-nitrophenyl-O-[beta]-lactoside (C16ampNPLac), was designed with an amide bond situated at a distance of two carbons from the glycosidic oxygen. Its susceptibility to the three endo-type glycosylceramidases was examined as shown in Table I. As expected, C16ampNPLac was digested by all three enzymes and the hydrolysis rates could be easily determined by spectrophotometric assay with no need for TLC analysis. C16ampNPLac was used as substrate to kinetically study EGCases and CGase and estimate the affinity of the substrate toward these enzymes. Lineweaver-Burk plot analysis of hydrolysis by EGCase/CGase showed high affinity toward the lactoside compared to GSL substrates (Ito and Yamagata, 1989; Zhou et al., 1989; Ashida et al., 1992).
Aglycone specificity of EGCase and CGase
Endo-type glycosylceramidases, capable of cleaving the glycosidic linkage between oligosaccharide and ceramide in GSLs, include EGCases from Rhodococcus sp. (Ito and Yamagata, 1986) and Corynebacterium sp. (Ashida et al., 1992) and CGases from leech (Li et al., 1986) and earthworm (Li et al., 1987). The present study demonstrates for the first time that endo-type glycosylceramidases may be classed as EGCase and CGase based on substrate specificity. These were shown to be accidentally but correctly named, based on their substrate specificity as shown in this study.
CnamEtLac was studied to determine whether the N-acylamido bond at the same site as in the ceramide is required for substrate recognition by endo-type glycosylceramidases. EGCases from Rhodococcus sp. and Corynebacterium sp. hydrolyzed CnamEtLac significantly more than n-alkyl lactosides. It follows then that at least the N-acyl structure in ceramide is partially responsible for substrate recognition by EGCases isolated from microorganisms. Lactosides with the N-acyl chain linked to a butylamino, CnamBuLac, instead of ethylamino group were not hydrolyzed by EGCases; so the action of EGCases is apparently affected by the distance between the amide bond and oligosaccharide unit, indicating that the lactose and N-acyl structures in GSLs are the minimal requirements for EGCase-catalyzed hydrolysis. On the other hand, CGase showed no structural preference for the position of the amide bond specified above but rather hydrophobic aglycone length and/or size may be essential for substrate recognition. This perhaps would explain why CGase and EGCase cleave not only synthetic alkyl lactosides but also lyso-GSL (Zhou et al., 1989; Ashida et al., 1995). The mechanisms for substrate recognition by EGCase and CGase are thus likely to differ and may account for the considerable transglycosylation activity of CGase (Li et al., 1991). This study suggests that, for the first time, the arbitrary designation of EGCase and CGase for the same catalytic activity now has a basis in the substrate specificity. This specificity may simply be the difference between prokaryotic and eukaryotic organisms. Further study on cDNA cloning or discovery the enzyme in mammals may answer this hypothesis.
Chromogenic substrate for both EGCase and CGase
Wang et al. have reported that CGases from leech and earthworm hydrolyze the synthetic substrate, 4-methylumbelliferyl 6[prime]-O-benzyl-[beta]-lactoside, the enzymes from Rhodococcus sp. and Corynebacterium sp. do not. (Wang et al., 1996). The present study also shows differences in substrate specificity between EGCase and CGase, providing a possible explanation for the above susceptibilities. But lactosides having ceramide-mimicking N-acyl chain were highly susceptible to all enzymes in this study. The C16ampNPLac, p-nitrophenyl lactoside derivative, possessing a hexadecanoyl amide separated by two carbons from the glycoside oxygen, served as a substrate for all endo-type glycosylceramidases. Km of CGase has been reported to be 15 µM for GM1, five times higher than that for LacCer (Zhou et al., 1989). Km of CGase for the chromogenic substrate, C16ampNPLac, was determined to be 28 µM, thus showing C16ampNPLac was a substrate as effective as GSLs from natural sources. The affinity of the substrate to endo-type glycosylceramidases shown in Table I appeared to be basically the same as that of natural GSLs despite the lower affinity of its sugar unit (lactose) toward the enzymes compared to the sugar moiety of GM1. Km of EGCases from Rhodococcus sp. and Corynebacterium sp for the chromogenic lactoside were 2.9 mM and 1.4 mM, respectively, comparable to those in the literature (Ito and Yamagata, 1989; Ashida et al., 1992).The chromogenic lactoside may be used in place of natural GSLs as a substrate and may facilitate the detection of EGCase or CGase activities in other systems.
Enzymes
EGCase from Rhodococcus sp. and EGCase-C from Corynebacterium sp. were purified as described previously (Ito and Yamagata, 1989; Ashida et al., 1992). CGase from leech was purchased from Boehringer Mannheim. Precoated Silica Gel 60 HPTLC plates were from E. Merck (Darmstadt, Germany). The standard lactosylceramide was kindly donated by SNOW BRAND Co. Ltd. (Japan).
Amphipathic lactosides
n-Alkyl lactosides were prepared according to Vill et al. (Vill et al., 1989). 2-N-Hexadecanoylamino-4-nitrophenyl-O-[beta]-lactoside (C16ampNPLac) and N-acyl-aminoethyl lactosides were prepared as described previously (Miura et al., 1996) as were also N-acyl-aminobutyl lactosides except that 4-amino-butanol was used instead of 2-amino-ethanol.
Enzyme assays
For assays with EGCase from Rhodococcus sp., the reaction mixture contained 1 mM lactoside and an appropriate amount of the enzyme in 50 µl 50 mM sodium acetate buffer, pH 5.0, containing 0.4% Triton X-100. For assays with EGCase from Corynebacterium sp. and CGase, the reactions were conducted as above except that 10 mM sodium acetate buffer, pH 6.5, containing 0.1% Triton X-100 and 50 mM sodium acetate buffer, pH 5.0, containing 2.5 µg/µl of sodium taurodeoxycholate, respectively, were used. On completion of the reactions, aliquots were subjected to HPTLC using 1-butyl alcohol/acetic acid/water (2:1:1, v/v) as the developing solvent. Lactose and lactosides were visualized by spraying the HPTLC plates with orcinol-sulfuric acid reagent and scanning with a Shimadzu CS-9000 chromatoscanner. The extent of hydrolysis was determined as described (Ito and Yamagata, 1989). For assay with C16ampNPLac, after enzymatic hydrolysis, the reaction mixture was treated with 50 µl 0.1 M glycine-NaOH buffer, pH 10.0, and 100 µl of ethanol to solubilize chromogenic product. Optical density at 410 nm due to hydrolysis was read following centrifugation. Released chromophore was determined using absorption coefficient of 12,500 at pH 10.
We thank Dr. Toshinori Sato at Tokyo Institute of Technology for his kind guidance in the synthesis of lactosides, and we are indebted to SNOW BRAND for providing standard LacCer. This work was supported in part by funds from the Mitsubishi Chemical Corporation and Seikagaku Kogyo Corporation, the Fujisawa Foundation, Life Science Foundation of Japan; grants-in-aid for scientific priority areas (7558092 and 09240104) from the Ministry of Education, Science, and Culture of Japan (T.Y.); and a Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists (Y.M.).
EGCase, endoglycoceramidase; CGase, ceramide glycanase; LacCer, lactosylceramide; GSL, glycosphingolipid; CnLac, n-alkyl [beta]-lactoside; CnamEtLac, N-acylaminoethyl [beta]-lactoside; CnamBuLac, N-acylaminobutyl [beta]-lactoside; CnamEt-S-Lac, N-acylaminoethyl thiolactosides; C16ampNPLac, 2-N-hexadecanoyl-amino-4-nitrophenyl-O-[beta]-lactoside; HPTLC, high performance thin-layer-chromatography.
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