Skip to main page content

• HOME
• Current Issue
• Archive
• Alerts
• About ASM
• Contact us
• Tech Support
• Journals.ASM.org

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Sumida, T. Articles by Ito, M. Search for Related Content PubMed PubMed Citation Articles by Sumida, T. Articles by Ito, M. Agricola Articles by Sumida, T. Articles by Ito, M.

 Previous Article  |  Next Article 

Applied and Environmental Microbiology, November 2002, p. 5241-5248, Vol. 68, No. 11
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.11.5241-5248.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Utilization of Ganglioside-Degrading Paenibacillus sp. Strain TS12 for Production of Glucosylceramide

Tomomi Sumida, Noriyuki Sueyoshi, and Makoto Ito^*

Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan

Received 1 April 2002/ Accepted 9 August 2002

Top Abstract Introduction Materials and Methods Results Discussion References

 
Gangliosides, sialic acid-containing glycosphingolipids, are membrane constituents of vertebrates and are known to have important roles in cellular differentiation, adhesion, and recognition. We report here the isolation of a bacterium capable of degrading gangliotetraose-series gangliosides and a new method for the production of glucosylceramide with this bacterium. GM1a ganglioside was found to be sequentially degraded by Paenibacillus sp. strain TS12, which was isolated from soil, as follows: GM1a asialo GM1 asialo GM2 lactosylceramide glucosylceramide. TS12 was found to produce a series of ganglioside-degrading enzymes, such as sialidases, ß-galactosidases, and ß-hexosaminidases. TS12 also produced ß-glucosidases, but glucosylceramide was somewhat resistant to the bacterial enzyme under the conditions used. Taking advantage of the specificity, we developed a new method for the production of glucosylceramide using TS12 as a biocatalyst. The method involves the conversion of crude bovine brain gangliosides to glucosylceramide by coculture with TS12 and purification of the product by chromatography with Wakogel C-300 HG.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Glycosphingolipids (GSLs) are amphipathic compounds consisting of a hydrophilic sugar moiety and a hydrophobic lipid moiety, ceramide (Cer). Gangliosides, which are GSLs that contain a sialic acid(s), are present mainly in the plasma membranes of vertebrates and are relatively abundant in the neuronal cells (18). Several lines of evidence indicate that gangliosides function as modulators for cellular differentiation, adhesion, and recognition (8, 32). There is also evidence that GSLs, including gangliosides, are enriched with cholesterol and GPI anchor proteins, form microdomains on the plasma membrane (9), and modulate inter- and intracellular signaling via interactions with various protein kinases, such as src family kinases (15), and cell surface receptors (8). On the other hand, gangliosides have been demonstrated to be receptors for microorganisms and their toxins (25).

Some microbes produce GSL-degrading enzymes, including sialidases (neuraminidase), which acts on the sialic acid residues of gangliosides (27); an endoglycoceramidase, which hydrolyzes the linkage between the oligosaccharide and Cer of various GSLs, including gangliosides (13); and a sphingolipid ceramide N-deacylase (SCDase), which hydrolyzes the N-acyl linkage of Cer in various GSLs, as well as sphingomyelin, to produce free fatty acids and the corresponding lyso-GSLs (12). However, these microbes hydrolyze a specific linkage of gangliosides, resulting in incomplete degradation of the substrates. It should be noted that there have been no reports of microbial exoglycosidases that are capable of degrading ganglio-series GSLs except sialidase.

GSLs are usually prepared in two ways: isolation from animal sources and synthesis by chemical methods. Moreover, methods in which a specific microbe is utilized are useful and promising for the production of GSLs on a large scale. For example, a sialidase-producing marine bacterium, Pseudomonas sp. strain YF-2, was successfully utilized for production of GM1a ganglioside as a microbial catalyst (7).

In this paper we describe the novel bacterium strain TS12, which sequentially decomposes gangliotetraose-series gangliosides by producing a series of exoglycosidases. We also describe a method in which TS12 is used as a microbial catalyst for the production of glucosylceramide (GlcCer), which is potentially important in axonal morphology and neuronal function (2, 16, 26).

Top Abstract Introduction Materials and Methods Results Discussion References

 
Materials.
Crude gangliosides were prepared from bovine brain as previously described (14). The following gangliosides and GSLs were purchased from Iatron Laboratories Inc. (Tokyo, Japan): GM3, GM2, GM1a, GD1a, GD1b, GT1b, GQ1b, globoside (Gb4Cer), and sulfatide. GlcCer and lactosylceramide (LacCer) were obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Precoated Silica Gel 60 thin-layer chromatography (TLC) plates were obtained from Merck (Darmstadt, Germany). Sodium taurodeoxycholate (TDC), 4-methylumbelliferyl-ß-D-glucoside, 4-methylumbelliferyl-ß-D-galactoside, 4-methylumbelliferyl-N-acetyl-ß-D-galactosaminide, and 4-methylumbelliferyl--D-N-acetylneuraminic acid were purchased from Sigma. 4-Nitrobenz-2-oxa-1,3-diazole (NBD)-labeled GM1a (NBD-C12:0) (see Fig. 3B) was enzymatically synthesized by using SCDase as described previously (21).

View larger version (20K): [in this window] [in a new window]

FIG. 3. Degradation of GM1a by TS12 (A) and fluorescence (NBD)-labeled GM1a (B).

Bacterial strains.
TS12 was isolated from soil in Fukuoka Prefecture, Japan, by enrichment culturing by using medium A (0.5% NaCl, 0.05% K₂HPO₄, 0.05% NH₄Cl, 0.05% TDC, 0.05% GM1a; pH 7.4). It was grown at 30°C in medium B (0.5% Polypeptone, 0.1% yeast extract, 0.2% NaCl, 0.05% TDC, 0.05% crude bovine brain gangliosides; pH 7.4). Paenibacillus alvei IFO 3343, P. amylolyticus IFO 13625, P. macerans IFO 15307, P. pabuli IFO 13638, P. polymyxa IFO 15309, and P. validus IFO 13636 were purchased from the Institute for Fermentation Osaka (Osaka, Japan) and were grown at 30°C in medium C (1% Polypeptone, 0.2% yeast extract, 0.1% MgSO₄ · 7H₂O, 0.05% TDC, 0.05% crude bovine brain gangliosides; pH 7.0). The medium was solidified with 1.8% agar if necessary.

Physiological and biochemical analysis of bacteria.
The general procedures used for identification of bacteria were conducted as described in Bergey's Manual of Determinative Bacteriology, 8th ed. (4). Motility, morphology, and Gram staining characteristics were determined by light microscopy. Kovács oxidase test was employed (17), and utilization of each carbohydrate was determined in Hugh-Leifson medium (11). Bacterial DNAs were extracted and purified by the method of Marmur (20), and guanine-plus-cytosine (G+C) contents were determined by high-performance liquid chromatography as described by Tamaoka and Komagata (30). A standard mixture of the four deoxyribonucleotides was purchased from Yamasa Shoyu (Chiba, Japan).

Phylogenetic analysis.
16S ribosomal DNA (rDNA) was amplified by using the universal primers p27f (5'-AGA GTT TGA TCM TGG CTC AG-3'; positions 8 to 27; Escherichia coli numbering [3]) and p1492r (5'-GGC TAC CTT GTT ACG ACT T-3'). PCR products were purified from a 1.0% agarose gel and sequenced directly by using eight different sequencing primers (10). The nucleotide sequences were aligned, and phylogenetic relationships were analyzed by using CLUSTAL W (31) and other 16S rDNA sequences obtained from the Ribosomal Database Project II (19).

Production of GlcCer from crude gangliosides with TS12.
An aliquot of TS12 cells from an agar slant culture was transferred into 5 ml of medium B and incubated at 30°C for 2 days with shaking. The culture was transferred into a 200-ml flask containing 50 ml of medium B and incubated at 30°C with shaking. After incubation at 30°C for 3 days, 5 volumes of chloroform-methanol (2/1, vol/vol) was added to the culture supernatant, shaken, and left to settle at room temperature. Then the lower layer was withdrawn, dried with a rotary evaporator, dissolved in a small amount of chloroform-methanol (2/1, vol/vol), and applied to a Wakogel C-300 HG column (0.7 by 35 cm). GlcCer and LacCer were eluted from the column with chloroform-methanol-water (90/10/0.5, vol/vol) and chloroform-methanol (2/1, vol/vol), respectively. The fractions containing GlcCer and LacCer were pooled separately and dried with the rotary evaporator.

TLC of GSLs.
Twenty microliters of each culture supernatant or effluent from the column was dried with a Speed Vac SC110 (Savant Instruments), dissolved in 10 µl of chloroform-methanol (2/1, vol/vol), and then applied to a Silica Gel 60 TLC plate, which was developed with solvent I (chloroform-methanol-0.02% aqueous CaCl₂, 5/4/1 [vol/vol/vol]) or solvent II (chloroform-methanol-25% ammonia, 90/20/0.5 [vol/vol/vol]). GSLs were visualized by spraying the plate with orcinol-H₂SO₄ reagent (29) and were quantified with a CS-9300PC chromatoscanner (Shimadzu, Kyoto, Japan) with the mode set at 540 nm. NBD-labeled substrates were visualized under a UV transilluminator. To examine the purity of the GlcCer obtained, 10 µg of sample was applied to a TLC plate which was developed with solvent II. GlcCer and contaminants were visualized by staining with Coomassie brilliant blue R-250 (22) and orcinol-H₂SO₄ reagents.

TOF-MS analysis of GlcCer.
For time of flight mass spectrometry (TOF-MS) analysis of GlcCer, equal amounts of sample (10 µM) dissolved in chloroform-methanol (2/1, vol/vol) and a matrix solution (10 mg of 2,5-dihydroxybenzoic acid per ml in a 1:1 mixture of methanol and water) were mixed, and 1 µl of the mixture was loaded onto a sample plate and allowed to dry. Then the sample plate was loaded into Voyager Vestec apparatus (PerSeptive Biosystem). A nitrogen laser (337 nm) was used for ionization.

Preparation of culture supernatant.
Aliquots of cells of TS12 and other bacterial strains were transferred into 5 ml of medium C and incubated at 30°C for 2 days with shaking. After incubation, each culture was centrifuged at approximately 17,000 x g to 20,000 x g for 5 min. To remove the bacterial cells completely, the supernatant was also passed through a 0.2-µm-pore-size filter (EB-DISK25; KANTO Chemical Co., Tokyo, Japan).

Glycosidase assay.
Exoglycosidase activity was measured by using various 4-methylumbelliferyl glycosides (4MU-glycosides) as substrates. Each reaction mixture contained 30 nmol of a 4MU-glycoside and 10 µl of culture supernatant in 100 µl of 20 mM sodium acetate buffer (pH 6.0). Following incubation at 37°C for 1 h, the reaction was stopped by adding 100 µl of 1 M glycine-NaOH buffer (pH 10), and the absorbance was measured at 325 nm. The control consisted of incubation without supernatant. One unit of enzyme was defined as the amount which catalyzed the release of 1 µmol of 4-methylumbelliferyl per min from 4MU-glycosides under the conditions described above.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Isolation and identification of a bacterium capable of degrading gangliosides.
The ganglioside-degrading bacterium TS12 was isolated from soil by enrichment culturing by using medium A. Strain TS12 is a short rod-shaped bacterium that is peritrichous (Fig. 1A). This bacterium was assigned to the genus Paenibacillus based on biochemical and physiological properties (Table 1) and 16S rDNA analysis data. However, phylogenetic analysis revealed that TS12 was not identical to any known type strain in the genus Paenibacillus (Fig. 1B).

View larger version (20K): [in this window] [in a new window]

FIG. 1. Electron micrograph and phylogenetic tree of TS12. (A) TS12 cell stained with uranium acetate and observed with an electron microscope (JEM-1010). (B) Phylogenetic tree. Partial 16S rDNA fragments were amplified by PCR with universal primers. The nucleotide sequences were determined and then subjected to a phylogenetic analysis by using CLUSTAL W and 16S rDNA sequences of other Paenibacillus sp. obtained from a DNA database. The DDBJ accession numbers for the sequences used for phylogenetic comparisons are as follows: P. alvei, AJ320491; P. pabuli, X60630; P. amylolyticus, P85496; P. larvae subsp. pulvifaciens, AY030080, P. validus, AF353697; P. macerans, X57306; and P. polymyxa, AJ320493. The phylogenetic tree was constructed by the neighbor-joining method (24). The details are described in Materials and Methods.

View this table: [in this window] [in a new window]

TABLE 1. Morphological, physiological, and biochemical properties of strain TS12

Degradation of GSLs during cultivation with TS12.
To clarify the metabolism of GM1a ganglioside by TS12, the strain was cultured in a synthetic liquid medium containing GM1a at 30°C with shaking. Twenty microliters of the culture supernatant was withdrawn every 6 h and analyzed by TLC (Fig. 2A). The degradation of GM1a and the generation of metabolites were quantified with a TLC chromatoscanner (Fig. 2B). The content of GM1a in the culture supernatant gradually decreased with time, and the content of asialo GM1 (sialic acid- removed GM1) increased. After 12 h, GM1a was converted to mainly asialo GM1, and then it was degraded to LacCer. Finally, LacCer was converted to GlcCer. We noted that the R_f of the GlcCer generated from GM1a did not completely correspond to that of standard GlcCer derived from human spleen (Fig. 2A). This may have been a consequence of the different composition of Cer, as described below. In conclusion, GM1a was converted to GlcCer by TS12 via the following putative pathway: GM1a asialo GM1 asialo GM2 LacCer GlcCer (Fig. 3A). The GlcCer content increased slowly, and GlcCer accumulated in the culture supernatant (Fig. 2A and B). These results may indicate that TS12 does not produce ß-glucosidase or that the TS12 ß-glucosidase does not hydrolyze GlcCer under the conditions used. We found that a more susceptible substrate, NBD-GM1a (NBD-C12:0), a fluorescent GM1a with a dodecanoyl group (Fig. 3B), was sequentially degraded by coculture with TS12 and was finally converted to NBD-Cer (Fig. 2C, lane 2), suggesting that TS12 produced ß-glucosidases capable of degrading NBD-GlcCer (NBD-C12:0). Meanwhile, the natural GlcCer with a long-chain fatty acyl group in the Cer was hardly degraded at all by the enzyme under the conditions used. Very recently, we cloned and characterized a TS12 ß-glucosidase (glucocerebrosidase), and the data revealed that the enzyme hydrolyzed NBD-GlcCer (NBD-C12:0) much faster than it hydrolyzed GlcCer containing stearic acid (C18:0) (28).

View larger version (55K): [in this window] [in a new window]

FIG. 2. Metabolic degradation of GM1a and NBD-GM1a during cultivation with TS12. TS12 cells were transferred into 250 µl of medium A and incubated at 30°C for different times. Twenty microliters of the culture supernatant was withdrawn every 6 h and analyzed by TLC with solvent I. (A) TLC stained with orcinol-H2SO4 reagent. AsGM1, asialo GM1; TDC, taurodeoxycholate. (B) Quantification of the compounds shown in panel A with a Shimadzu CS-9300PC chromatoscanner with the reflectance mode set at 540 nm. The values are means ± standard deviations for triplicate determinations. (C) Degradation of NBD-GM1a. One hundred picomoles of fluorescent GM1a was incubated with TS12 at 30°C for 3 days in medium A. The reaction product was separated by TLC by using solvent I. Lane 1, standard NBD-GM1a; lane 2, NBD-GM1a plus TS12; lane 3, standard NBD-GlcCer (NBD-C12:0); lane 4, standard NBD-Cer (NBD-C12:0).

Production of glycosidases involved in ganglioside degradation by TS12.
TS12 degraded GM1a to produce GlcCer when the strain was cultured with the ganglioside. Thus, we examined whether TS12 produces glycosidases involved in the degradation of GM1a. First, we examined the degradation of NBD-GM1a by a TS12 cell-free culture supernatant. NBD-GM1a was sequentially degraded by the cell-free supernatant after incubation for 12 h to generate GlcCer (Fig. 4A, lane 2), part of which was converted to NBD-Cer after incubation for 24 h (Fig. 4B, lane 2), indicating that TS12 produced a series of exoglycosidases required for degradation of NBD-GM1a to NBD-Cer. In contrast, NBD-GM1a was not hydrolyzed by the cell-free supernatants from six type strains of Paenibacillus spp. (Fig. 4A, lanes 5 to 10). Next, exoglycosidase activities in the cell-free supernatants of cultures of TS12 and type strains were examined by using various 4MU-glycosides. In the TS12 cell-free supernatant, four exoglycosidases required for the degradation of GM1a were detected, while no type strain produced sialidase and ß-N-acetylgalactosaminidase, resulting in no degradation of GM1a (Table 2).

View larger version (35K): [in this window] [in a new window]

FIG. 4. Degradation of NBD-GM1a by cell-free supernatant of TS12. NBD-GM1a (100 pmol) was incubated at 37°C for different times with 20 µl of cell-free supernatant of TS12 or another strain. The reaction products were analyzed by TLC as described in Materials and Methods. (A) Incubation for 12 h. Lane 1, standard NBD-GM1a (NBD-C12:0); lane 2, NBD-GM1a with TS12 cell-free supernatant; lane 3, standard NBD-GlcCer (NBD-C12:0); lane 4, standard NBD-Cer (NBD-C12:0); lanes 5 to 10, NBD-GM1a with cell-free supernatants from Paenibacillus strains (lane 5, P. alvei; lane 6, P. amylolyticus; lane 7, P. validus; lane 8, P. pabuli; lane 9, P. macerans; lane 10, P. polymyxa). AsGM2, asialo GM2. (B) Incubation for 24 h. Lane 1, standard NBD-GM1a; lane 2, NBD-GM1a with TS12 cell-free supernatant; lane 3, standard NBD-GlcCer; lane 4, standard NBD-Cer.

View this table: [in this window] [in a new window]

TABLE 2. Glycosidase activities of Paenibacillus sppa

Degradation of various GSLs by TS12.
It was found that TS12 metabolized GM1a by secreting a series of exoglycosidases required for GM1a degradation. Thus, we examined the degradation of various GSLs by TS12. After incubation at 30°C for 12 h, GT1b, GD1b, GD1a, GM1a, GM2, and GM3 were degraded completely, generating LacCer and GlcCer. LacCer was also degraded by TS12, but the process was slow and incomplete. No hydrolysis of Gb4Cer and sulfatide was observed under the conditions used (Table 3).

View this table: [in this window] [in a new window]

TABLE 3. Degradation of GSLs by TS12a

Production of GlcCer by TS12 and purification of GlcCer.
Since TS12 converts GM1a, GD1a, and GD1b, which are major gangliosides of bovine brains, to GlcCer under the conditions used, we devised a new method to prepare GlcCer from crude bovine brain gangliosides (mainly a mixture of GM1a, GD1a, and GD1b) using TS12 as a microbial catalyst. This method consisted of (i) conversion of crude gangliosides to GlcCer by culturing with TS12 and (ii) purification of GlcCer from the TS12 culture supernatant by Wakogel C-300 HG column chromatography. Crude bovine brain gangliosides (25 mg) were cultured with TS12 in 50 ml of liquid medium B at 30°C for 3 days, and the culture supernatant was withdrawn to examine the GSLs by TLC. We found that the major GSL in the supernatant was GlcCer, followed by LacCer. To separate GlcCer from LacCer, GSLs were extracted from the culture supernatant with 5 volumes of chloroform-methanol (2/1, vol/vol) and subjected to chromatography on a Wakogel C-300 HG column. GlcCer (fractions 9 to 15) was found to be clearly separated from LacCer (fractions 27 to 29) and other contaminants (fractions 5 and 31 to 33) (Fig. 5). Finally, 6.76 mg of GlcCer was obtained from 25 mg of crude gangliosides, so the yield was about 56.5% (if all GSLs in the starting material were considered to be GM1a). GlcCer was then subjected to a purity analysis. Figure 6 shows the location and purity of GlcCer on a TLC plate developed with solvent II after the preparation was stained with either orcinol-H₂SO₄ reagent (for carbohydrate-containing products) (Fig. 6A) or Coomassie brilliant blue reagent (for lipid-containing products) (Fig. 6B). The R_f of GlcCer obtained in this study (Fig. 6, lanes 1) almost, but not quite, corresponded to that of standard GlcCer derived from human spleen (Fig. 6, lanes 2). This may have been a consequence of the different composition of Cer, as described below. No contamination was detected in the prepared GlcCer (Fig. 6). Two bands for the GlcCer standard may have arisen because of a difference in the Cer moiety (6). The prepared GlcCer was analyzed further by TOF-MS in the positive-ion mode. The characteristic pseudomolecular [M+Na]⁺ ions were found at m/z 750.8 and m/z 778.9, which corresponded to two molecular species of GlcCer with different Cer types (Fig. 7). One type of Cer consisted of the sphingosine of the d18:1 long-chain base and the fatty acid of C18:0 stearic acid (molecular weight, 565). The other type consisted of the eicosasphingosine of d20:1 and the fatty acid of C18:0 stearic acid (molecular weight, 593). Relatively weak signals were found at m/z 728.6 and m/z 756.9, which corresponded to the respective [M+H]⁺ ions. The Cer composition of the prepared GlcCer was exactly the same as that of bovine brain GM1a (1) but was somewhat different from that of human spleen GlcCer (6). This explains the difference in the R_f values of the prepared GlcCer (from bovine brain) and the standard GlcCer (from human spleen).

View larger version (25K): [in this window] [in a new window]

FIG. 5. Purification of GlcCer from culture supernatant of Paenibacillus sp. strain TS12. Crude bovine brain gangliosides (25 mg) were incubated at 30°C for 3 days with TS12 in 50 ml of medium B. GlcCer was isolated from the TS12 culture supernatant by using a Wakogel C-300 HG column. The elutes were checked by TLC as described in Materials and Methods. Std, standard containing LacCer (lower band) and GlcCer (upper band).

View larger version (42K): [in this window] [in a new window]

FIG. 6. Purity of the prepared GlcCer. Aliquots of the purified GlcCer and standard GlcCer (10 µg each) were analyzed by TLC by using solvent II and were visualized with orcinol-H2SO4 (29) (A) or Coomassie brilliant blue R-250 (22) (B). Lane 1, purified GlcCer; lane 2, standard GlcCer from spleens of patients with Gaucher disease.

View larger version (15K): [in this window] [in a new window]

FIG. 7. TOF-MS analysis of the purified GlcCer. The analysis was conducted with a Voyager Vestec mass spectrometer (PerSeptive Biosystem) in the positive-ion mode with 2,5-dihydroxybenzoic acid as the matrix.

In summary, we concluded that GlcCer was produced from crude bovine brain gangliosides by sequential hydrolysis of the sugar chains with TS12 exoglycosidases.

Top Abstract Introduction Materials and Methods Results Discussion References

 
In vertebrates, invertebrates, plants, and some microbes GSLs are constituents of plasma membranes. These GSLs should be degraded by microbes in natural habitats like other biological compounds, such as polysaccharides, lipids, and proteins. If they are not, GSLs should accumulate on earth, but this is unlikely. However, there have been very few reports of metabolic degradation of GSLs by microbes (12, 13, 27). This paper provides the first report of metabolic degradation of ganglio-series GSLs that generates GlcCer by culture with a single strain of bacteria. TS12 seems to possess all the exoglycosidases required for degradation of gangliotetraose-series gangliosides. Interestingly, TS12 is likely to utilize GSL-derived monosaccharides as a carbon source, because no monosaccharides were detected by TLC in medium A after degradation of GM1a by TS12 (data not shown). TS12 was isolated from soil, and thus it is possible that in nature gangliosides are metabolized by such bacteria or by combinations of several microbes capable of producing GSL-degrading enzymes. It is interesting that human fecal bacteria, including Ruminococcus sp. and Bifidobacterium sp., produce exoglycosidases which degrade lacto-series GSLs but not ganglio-series GSLs, such as GM1a (5).

The following metabolic pathway for degradation of GM1a in human lysosomes has been proposed: GM1a GM2 GM3 LacCer GlcCer (23). Meanwhile, GM1a was metabolized in the TS12 culture as follows: GM1a asialo GM1 asialo GM2 LacCer GlcCer (Fig. 3A). The difference may result from the specificity of ß-hexosaminidase (ß-N-acetylgalactosaminidase); i.e., the human enzyme hydrolyzes the ß1-4 N-acetyl-ß-D-galactosaminide linkage even if the neighboring Gal is linked with N-acetylneuraminic acid, while the TS12 enzyme does not.

Although TS12 was assigned to the genus Paenibacillus based on biochemical and physiological properties and 16S rDNA analysis results, TS12 was not identical to any Paenibacillus sp. type culture. Furthermore, it was found that no type culture of Paenibacillus tested was able to degrade GM1a, suggesting that TS12 could be a new species of the genus Paenibacillus.

GlcCer has important roles in axonal morphology and neuronal function (2, 16, 26). Among GSLs, GlcCer is a minor constituent of normal tissues, and thus it is prepared from spleens of patients with Gaucher disease. On the other hand, GSLs belonging to the gangliotetoraose family are relatively abundant in bovine brain, and it is much easier to obtain large amounts of these compounds. In this paper we describe a new method that involves the gangliotetraose GSL-degrading bacterium TS12 as a microbial biocatalyst and bovine brain gangliosides as the starting material. This method is based on the observation that TS12 produces various exoglycosidases required for the conversion of GM1a to GlcCer, and the final product is somewhat resistant to the TS12 ß-glucosidases, resulting in the accumulation of GlcCer in cultures of TS12. The method described here is suitable for preparation of large quantities of GlcCer due to its high yield and low cost and should be applicable at the laboratory scale (milligram level) and the industrial scale (kilogram level).

 
We thank H. Higashi of the Mitsubishi Kagaku Institute of Life Sciences (Japan) and M. Suzuki of RIKEN (Japan) for performing the TOF-MS analysis of GlcCer.

This work was supported in part by grant-in-aid for scientific research (B) 13460044.

 
* Corresponding author. Mailing address: Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan. Phone: 81-92-642-2898. Fax: 81-92-642-2898 or 81-92-642-2907. E-mail: makotoi{at}agr.kyushu-u.ac.jp.

Top Abstract Introduction Materials and Methods Results Discussion References

 

1
1. Arita, M., M. Iwamori, T. Higuchi, and Y. Nagai. 1983. Negative ion fast atom bombardment mass spectrometry of gangliosides and asialo gangliosides: a useful method for the structural elucidation of gangliosides and related neutral glycosphingolipids. J. Biochem. (Tokyo) 94:249-256.[Abstract/Free Full Text]
2
2. Boldin, S. A., and A. H. Futerman. 2000. Up-regulation of glucosylceramide synthesis upon stimulation of axonal growth by basic fibroblast growth factor. J. Biol. Chem. 275:9905-9909.[Abstract/Free Full Text]
3
3. Brosius, J., M. L. Palmer, P. J. Kennedy, and H. F. Noller. 1978. Complete nucleotide sequence of a 16S ribosomal RNA gene from Escherichia coli. Proc. Natl. Acad. Sci. USA 75:4801-4805.[Abstract/Free Full Text]
4
4. Buchanan, R. E., and N. E. Gibbons (ed.). 1974. Bergey's manual of determinative bacteriology, 8th ed. The Williams & Wilkins Co., Baltimore, Md.
5
5. Falk, P., L. C. Hoskins, and G. Larson. 1990. Bacteria of human intestinal microflora produce glycosidases specific for lacto-series glycosphingolipids. J. Biochem. (Tokyo) 108:466-474.[Abstract/Free Full Text]
6
6. Fujiwaki, T., S. Yamaguchi, K. Sukegawa, and T. Taketomi. 1999. Application of delayed extraction matrix-assisted laser desorption ionization time-of-flight mass spectrometry for analysis of sphingolipids in tissues from sphingolipidosis patients. J. Chromatogr. B 731:45-52.[CrossRef]
7
7. Fukano, Y., and M. Ito. 1997. Preparation of GM1 ganglioside with sialidase-producing marine bacteria as a microbial biocatalyst. Appl. Environ. Microbiol. 63:1861-1865.[Abstract]
8
8. Hakomori, S., K. Handa, K. Iwabuchi, S. Yamamura, and A. Prinetti. 1998. New insights in glycosphingolipid function: 'glycosphingolipid domain' a cell surface assembly of glycosphingolipids with signal transducer molecules, involved in cell adhesion coupled with signaling. Glycobiology 8:xi-xix.
9
9. Harder, T., and K. Simons. 1997. Caveolae, DIGs, and the dynamics of sphingolipid-cholesterol microdomains. Curr. Opin. Cell Biol. 9:534-542.[CrossRef][Medline]
10
10. Hiraishi, A. 1992. Direct automated sequencing of 16S rDNA amplified by polymerase chain reaction from bacterial cultures without DNA purification. Lett. Appl. Microbiol. 15:210-213.[Medline]
11
11. Hugh, R., and E. Leifson. 1953. The taxonomic significance of fermentative versus oxidative metabolism of carbohydrates by various gram-negative bacteria. J. Bacteriol. 66:24-26.[Free Full Text]
12
12. Ito, M., T. Kurita, and K. Kita. 1995. A novel enzyme that cleaves the N-acyl linkage of ceramides in various glycosphingolipids as well as sphingomyelin to produce their lyso forms. J. Biol. Chem. 270:24370-24374.[Abstract/Free Full Text]
13
13. Ito, M., and T. Yamagata. 1986. A novel glycosphingolipid-degrading enzyme cleaves the linkage between the oligosaccharide and ceramide of neutral and acidic glycosphingolipids. J. Biol. Chem. 261:14278-14282.[Abstract/Free Full Text]
14
14. Kanfer, J. N. 1969. Preparation of ganglioside. Methods Enzymol. 14:660-664.
15
15. Kasahara, K., K. Watanabe, K. Takeuchi, H. Kaneko, A. Oohira, T. Yamamoto, and Y. Sannai. 2000. Involvement of gangliosides in glycosylphosphatidylinositol-anchored neuronal cell adhesion molecule TAG-1 signaling in lipid raft. J. Biol. Chem. 275:34701-34709.[Abstract/Free Full Text]
16
16. Korkotian, E., A. Schwarz, D. Pelled, G. Schwarzmann, M. Segal, and A. H. Futerman. 1999. Elevation of intracellular glucosylceramide levels results in an increase in endoplasmic reticulum density and in functional calcium stores in cultured neurons. J. Biol. Chem. 274:21673-21678.[Abstract/Free Full Text]
17
17. Kovacs, N. 1956. Identification of Pseudomonas pyocyanea by the oxidase reaction. Nature 178:703.[Medline]
18
18. Ledeen, R. W. 1989. Biosynthesis, metabolism, and biological effects of gangliosides, p. 43-83. In R. U. Margolis and R. K. Margolis (ed.), Neurobiology of glycoconjugates. Plenum Press, New York, N.Y.
19
19. Maidak, B. L., J. R. Cole, C. T. Parker, Jr., G. M. Garrity, N. Larsen, B. Li, T. G. Lilburn, M. J. McCaughey, G. J. Olsen, R. Overbeek, S. Pramanik, T. M. Schmidt, J. M. Tiedje, and C. R. Woese. 1999. A new version of the RDP (Ribosomal Database Project). Nucleic Acids Res. 27:171-173.[Abstract/Free Full Text]
20
20. Marmur, J. 1961. A procedure for the isolation of deoxyribonucleic acid from micro-organisms. J. Mol. Biol. 3:208-218.
21
21. Nakagawa, T., M. Tani, K. Kita, and M. Ito. 1999. Preparation of fluorescence-labeled GM1 and sphingomyelin by the reverse hydrolysis reaction of sphingolipid ceramide N-deacylase as substrates for assay of sphingolipid-degrading enzymes and for detection of sphingolipid-binding proteins. J. Biochem. (Tokyo) 126:604-611.[Abstract/Free Full Text]
22
22. Nakamura, K., and S. Handa. 1984. Coomassie brilliant blue staining of lipids on thin-layer plates. Anal. Biochem. 142:406-410.[CrossRef][Medline]
23
23. Pshezhetsky, A. V., and M. Asmarina. 2001. Lysosomal multienzyme complex: biochemistry, genetics, and molecular pathophysiology. Prog. Nucleic Acids Res. Mol. Biol. 69:81-114.
24
24. Saitou, N., and M. Nei. 1987. The neighbor joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4:406-425.[Abstract]
25
25. Schengrund, C.-L., and N. J. Ringler. 1989. Binding of Vibrio cholerae toxin and heat-labile enterotoxin of Escherichia coli to GM1, derivatives of GM1, and nonlipid oligosaccharide polyvalent ligands. J. Biol. Chem. 264:13233-13237.[Abstract/Free Full Text]
26
26. Schwarz, A., and A. H. Futerman. 1997. Distinct roles for ceramide and glucosylceramide at different stages of neuronal growth. J. Neurochem. 17:2929-2938.
27
27. Sugano, K., M. Saito, and Y. Nagai. 1978. Susceptibility of ganglioside GM1 to a new bacterial neuraminidase, FEBS Lett. 89:321-325.
28
28. Sumida, T., N. Sueyoshi, and M. Ito. 2002. Molecular cloning and characterization of a novel glucocerebrosidase of Paenibacillus sp. TS12. J. Biochem. 132:237-243.
29
29. Svennerholm, L. 1956. The quantitative estimation of cerebrosides in nervous tissue. J. Neurochem. 1:42-53.[CrossRef][Medline]
30
30. Tamaoka, J., and K. Komagata. 1984. Determination of DNA base composition by reverse-phase high-performance liquid chromatography. FEMS Microbiol. Lett. 25:125-128.
31
31. Thompson, J. D., D. G. Higgins, and T. J. Gibson. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22:4673-4680.[Abstract/Free Full Text]
32
32. Wu, G., and R. W. Ledeen. 1991. Stimulation of neurite outgrowth in neuroblastoma cells by neuraminidase: putative role of GM1 ganglioside in differentiation. J. Neurochem. 56:95-104.[CrossRef][Medline]

---

Applied and Environmental Microbiology, November 2002, p. 5241-5248, Vol. 68, No. 11
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.11.5241-5248.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Sumida, T. Articles by Ito, M. Search for Related Content PubMed PubMed Citation Articles by Sumida, T. Articles by Ito, M. Agricola Articles by Sumida, T. Articles by Ito, M.