3Department of Life Sciences (Chemistry), Graduate School of Arts and Sciences, The University of Tokyo, Komaba, Meguro-ku, Tokyo 1538902, Japan, 4Division of Cell and Molecular Pathology, Department of Pathology, University of Zürich, 8091 Zürich, Switzerland, and the 5Pharmaceuticals Group, Nippon Kayaku Co., Ltd., Kita-ku Tokyo 1150042, Japan
Received on April 10, 2000; revised on July 25, 2000; accepted on July 31, 2000.
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Abstract |
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Key words: 1,5-anhydrofructose/-1,4-glucan lyase/glucosidase II/glycogenolytic pathway
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Introduction |
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In fungi (Yu et al., 1997) and red algae (Yu and Pedersen, 1993
; Yu et al., 1993
, 1995), several exo
-1,4-glucan lyases have been isolated, while the lyases in mammalian cells and organs still remain putative. The analysis of the amino acid sequences of the lyases of fungi and red algae revealed a high degree of conservation among the members in respective subfamilies (Bojsen et al., 1999a
,b). However, homology between fungi and algae was only 2528%. Remarkably, sequence alignments of lyase also demonstrated 2328% homology to the glycoside hydrolase family 31 (Yu et al., 1999
). This implies a common origin for these two enzyme families, although they differ in their catalytic mechanisms. In contrast to the glucoside hydrolase, the exo
-1,4-glucan lyase cleaves
-1,4-glucosidic bonds directly, without addition of water (Yu and Pedersen, 1993
).
In the present study, we have attempted to purify and characterize the putative mammalian exo -1,4-glucan lyase from rat liver. The partially determined amino acid sequences of the purified enzyme showed identity with corresponding segments of glucosidase II. We conversely found that
-1,3-glucosidase II from rat liver and CHO cells possessed a minor lyase activity. In the
-1,3-glucosidase II-deficient mouse lymphoma cell line PHAR2.7 and in glucosidase II-deficient Saccharomyces cerevisiae YG427, no lyase activity was detectable. This is the first report demonstrating two enzymatic activities for glucosidase II.
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Results |
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Discussion |
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In our attempts to purify -1,4-glucan lyase, we observed distinct lyase activities in rat liver homogenate. After DEAE Cellulose column chromatography, two lyase activities were found and in the present study, we have focussed on the enzyme activity bound to the column. Further studies will aim at the characterization of the enzyme activity which was not retained onto DEAE cellulose. While the DEAE-bound protein was reactive with concanavalin A, the unbound protein was unreactive (unpublished observations). From these data we concluded that the two distinct lyase activities correspond to different enzymes and the occurrence of 1,5-AnFru in mammalian cells (Kametani et al., 1996b
; Suzuki et al., 1996
) may be attributed to different enzymes. The seven N-terminal amino acid residues of the purified 110 kDa protein exhibiting lyase activity were identical with the amino acid residues 3340 in glucosidase II from human, pig, and mouse. In accordance, the N-terminal amino acid sequence of purified rat and pig liver glucosidase II (Trombetta et al., 1996
; Flura et al., 1997
; Hentges and Bause, 1997
) started at the same position suggesting that cleavage occurs at this specific site. The amino acid sequence of the 68 kDa protein was identical with an internal sequence of pig liver glucosidase II starting from residue 340, indicating that the 68 kDa is a degradation product which occurred during the purification. These data show that the purified protein exhibiting lyase activity is attributed to glucosidase II.
Glucosidase II removes the two glucose residues from the (Glc)2(Man)9(GlcNAc)2 oligosaccharide (Kornfeld and Kornfeld, 1985; Moremen et al., 1994
), and in liver it is a resident protein of the endoplasmic reticulum (Lucocq et al., 1986
). It is a ubiquitous enzyme and has been purified from various organs of different species (Burns and Touster, 1982
; Brada and Dubach, 1984
; Hino and Rothman, 1985
; Trombetta et al., 1996
; Hentges and Bause, 1997
). We demonstrate here that the purified glucosidase II, which is a tetrameric glycoprotein consisting of 110 kDa subunits, is immunoreactive toward anti-pig liver glucosidase II and has a lyase activity using maltose as substrate. Furthermore, CHO cells overexpressing pig liver glucosidase II showed a 1.5- to 2-fold enhanced lyase activity compared to nontransfected cells. On the other hand, glucosidase II deficient yeast or mouse lymphoma cells showed no lyase activity. These two established glucosidase II knock out strains lack both immunological and catalytic activities of glucosidase II. Concerning mutant mouse lymphoma PHAR 2.7 cell, Northern blot, and Western blot analysis show the absence of mRNA encoding glucosidase II and expression of the protein, respectively (Flura et al., 1997
). Construction of glucosidase II deficient yeast YG427 was performed by homologous recombination based on the sequence of pig liver glucosidase II (Jakob et al., 1998
). Collectively, these data strongly indicate that glucosidase II possesses two different enzymatic activities whereby the lyase activity is the minor activity and is detectable in various species.
We noticed that glucosidase II purified from rat liver, or from the homogenate of wild type yeast and BW5147 cells showed lyase activity only toward maltose but not toward glycogen. On the other hand, a crude hepatic preparation can produce 1,5-AnFru from glycogen (Kametani et al., 1996b). These data suggested that either some other not yet identified enzymes or amylases that can produce malto-oligosaccharides participate in this glycogenolytic pathway.
The mechanism for the production of 1,5-AnFru by glucosidase II in mammals so far is unknown. Concerning the catalytic reactions of glucosidase II, several reports have been presented (Alonso et al., 1991). Brada and Dubach demonstrated that glucosidase II cleaves not only
-1,3-bonds but also
-1,4-bonds (Brada and Dubach, 1984
). Based on the shared substrate and inhibitor specificity and the type of bond cleaved, one might speculate that two different enzymatic reactions, hydrolysis and elimination, take place at the same catalytic site forming a glucosyl-enzyme intermediate. The glucosyl moiety in the enzyme substrate complex may be released either hydrolytically or by elimination reaction primed by a different proton release mechanism (Yu et al., 1999
). The idea of dual ways for glucosyl intermediate dissociation is reminiscent of the fact that several
-glucosidases catalyze hydration of glucal to 2-deoxyglucose. The initial step of this reaction to the enzyme-substrate complex may be the reversal of dissociation of the glycosyl-enzyme intermediate through elimination reaction (note that 1,5-AnFru is a tautomer of 2-hydroxyglucal). We failed to demonstrate the hydration of 1,5-AnFru to glucose by
-glucosidases. This seems reasonable because 1,5-AnFru is stabilized in its hydrated form (Kametani et al., 1996a
) and seems to hardly undergo tautomeric isomerization to 2-hydroxyglucal in aqueous solution. The
-glucosidase-catalyzed hydration of glucal indicates that the enzyme has sufficient affinity to the unsaturated sugar. It is therefore anticipated that the structure of the 2,3-dihyropyran ring in glucal and 2-hydroxyglucal resemble the conformation of the backbone of the six-membered ring of the glucosyl moiety in the transition state on the enzyme.
Do all -glucosidases produce 1,5-AnFru? Among those investigated,
-glucosidase II is the only glucosidase which produced 1,5-AnFru as a side product. Neither a recombinant
-glucosidase (maltase, EC 3.2.1.20) of E.coli origin nor
-amylase from Aspergillus niger produced detectable amounts of 1,5-AnFru. Therefore, we tentatively conclude that only a few members of the glucoside hydrolase family produce 1,5-AnFru. The absence of 1,5-AnFru production in glucosidase II-deficient S.cerevisiae and PHAR2.7 cell also suggests that glucosidase II may be the only enzyme in these cells which is able to produce 1,5-AnFru.
In comparison with the major glycogenolytic pathway, phosphorolysis, which is catalyzed by phosphorylase and provides every organ with a sufficient amount of glucose as the energy source (Bollen et al., 1998), the activity of this anhydrosugar production is low in mammalian organs and is readily reduced to 1,5-AnGlc-ol. The physiological role of the production of 1,5-AnFru and 1,5-AnGlc-ol in mammalian cells is still unknown. Recently, it was demonstrated that 1,5-AnGlc-ol promotes glycogenolysis in E.coli (Shiga et al., 1999
), and therefore one may expect that they have also some effects on glycogen metabolism in mammalian organs.
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Materials and methods |
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The mouse lymphoma cell line BW5147 (glucosidase II-positive) and PHAR2.7 (glucosidase II-deficient) were kindly provided by Dr. I.Trowbridge (Salk Institute, San Diego, CA). Wild type CHO-K1 cells and CHO cells overexpressing pig liver glucosidase II (Flura et al., 1997) were cultured as described previously (Flura et al., 1997
). BW5147 and PHAR2.7 cell lines were grown in Dulbeccos modified Eagles medium containing 10% fetal bovine serum (FBS), penicillin (100 U/ml), and streptomycin (100 µg/ml) at 37°C and 10% CO2. CHO-K1 cells were grown in Hams F12 medium containing 10% FBS and CHO-K1 cells transfected with pig liver glucosidase II were grown in
MEM containing 10% FBS and G418 (1 mg/ml) at 37°C and 5% CO2. The following yeast strains were used: SS328 as glucosidase IIpositive wild type strain (Vijayraghavan et al., 1989
) and YG427 as glucosidase IIdeficient strain (Jakob et al., 1998
).
Purification of rat liver -1,4-glucan lyase
All steps were performed at 4°C. Frozen rat liver (200 g) were homogenized in 3 volumes of buffer A (20 mM sodium phosphate buffer, pH 7.0) containing 1.6 mM DTT. The cell debris, nuclei, mitochondria and lysosomes were removed by centrifugation at 38,000 x g for 20 min. The supernatant was acidified to pH 5.0 and left on ice for 30 min. Afterwards, the lysate was brought back to neutral pH and denatured proteins were removed. The supernatant was applied onto a DE 52 column (6 x 20 cm) equilibrated with buffer A. After washing with 2 volumes of buffer A, the proteins were eluted with a linear gradient of 00.5 M sodium chloride in buffer A. Two peaks of lyase activity, one in the flow through fractions and the other eluted with 0.20.25 M of sodium chloride, were obtained. The eluted fractions exhibiting lyase activity were concentrated by ultrafiltration (Amicon YM 30 membrane) and sodium sulfate was added to a final concentration of 1 M. Then the sample was loaded onto an Octyl Sepharose CL-4B column (5 x 15 cm) equilibrated with buffer A containing 1 M sodium sulfate. The column was washed with 2 volumes of this buffer and the proteins were eluted with buffer A. The eluted proteins were concentrated by ultrafiltration and rechromatographed on a DE 52 column (2.6 x 35 cm) using buffer A with the same sodium chloride gradient as described above. The fractions exhibiting lyase activity were concentrated and sodium sulfate was added to a final concentration of 1.5 M, then loaded onto Phenyl Superose column equilibrated with buffer A containing 1.5 M sodium sulfate. The column was washed with 2 volumes of this buffer and proteins were eluted by a decreasing linear salt gradient from 1.5 M to 0 M in buffer A. The peak fractions of phenyl Superose were diluted by adding 2 volumes of buffer B (20 mM sodium phosphate buffer, pH 7.8) and applied on a Mono Q column equilibrated with buffer B. Proteins were eluted with a linear gradient of 01 M sodium chloride in buffer B. The fractions exhibiting lyase activity were pooled and analyzed on a 6% SDSpolyacrylamide gel according to Laemmli (Laemmli, 1970). The gels were either silver stained or transferred to PVDF membrane (Towbin et al., 1979
) for amino acid sequencing. Amino acid sequences were determined by using the protein sequencer system Procise Model 492 from PE Biosystems (Foster City, USA).
Purification of rat liver glucosidase II
Rat liver glucosidase II was purified as described previously (Brada and Dubach, 1984). Briefly, microsomes were isolated from 100 g of frozen rat liver and proteins were extracted by the addition of 1% of Triton X-100. Glucosidase II was further purified on Fast Flow Q Sepharose, concanavalin A Sepharose, Mono Q, Sephacryl S-300, and phenyl Superose column chromatography, successively.
Measurement of enzymatic activity
1,5-AnFru activity was measured as described previously (Kametani et al., 1996a,b; Shiga et al., 1999
). The reaction mixture (200 µl), which contained maltose (2 mg/ml) as substrate, was incubated at 37°C for various time periods. The reaction was stopped by the addition of 4 volumes of ethanol. As internal standard, 20 ng of 13C6-1,5-AnFru was added, and denatured proteins were removed by centrifugation. After drying the supernatant, 160 µl of water, 20 µl of 10% O-ethylhydroxylamine-HCl and 20 µl of 0.5 M TrisHCl (pH 8.5) containing 1 mM EDTA were added to the sample and incubated at 80°C for 15 min. The resulting oximes were separated by a reverse-phase column (DIA-CHROMA ODS, 4.6 x 15 cm, Kakokishouji, Kawasaki) mounted on HPLC (LC-10, Shimadzu). The 1,5-AnFru ethyloxime was collected, dried, acetylated, and then injected onto GC-MS (Hewlett Packard HP 6890 GC System and 5973 Mass Selective Detector). The ion fragments of 1,5-AnFru (m/z = 169 and 211) and 13C6-1,5-AnFru (m/z = 175 and 217) were monitored.
Glucosidase II activity was measured using 0.1 mM 4-methylumbelliferyl -D-glucopyranoside as substrate (Brada and Dubach, 1984
). The amount of released methylumbelliferone was measured using a fluorescence photometer (excitation 360 nm, emission 450 nm).
The amount of glucose production was measured using a glucose assay kit.
Isoelectric focusing
The fractions exhibiting lyase activity were diluted with 3 volumes of 25 mM imidazole-HCl buffer (pH 7.4) and applied onto a Mono P column equilibrated with this buffer. The bound proteins were eluted with a linear gradient from pH 7.4 to 4.0 using polybuffer 74/HCl (pH 4.0) according to the manufacturers instructions.
Western blot analysis using anti-glucosidase II antibody
Purified glucosidase II or CHO cell homogenates were separated in a 7.5% SDSpolyacrylamide gel followed by transfer on nitrocellulose membrane using a semi-dry blotting apparatus (Towbin et al., 1979). The membrane was blocked with PBS containing 1% defatted milk powder and 0.05% Tween 20 for 1 h at room temperature and incubated with 1 µg/ml of anti-pig glucosidase II antibody overnight at 4°C, followed by alkaline phosphatase-conjugated goat anti-rabbit IgG antibodies. Color reaction was performed using nitro blue tetrazolium/BCIP as substrate.
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Footnotes |
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2 To whom correspondence should be addressed
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References |
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