2Laboratoire de Recherches sur les Macromolécules, UMR 7540 CNRS, Université Paris 13, avenue J.-B. Clément, 93430 Villetaneuse, France; 3Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, UMR 8601 CNRS, Université René Descartes, 45 rue des Saints-Pères, 75270 Paris, France; and 4Laboratoire de Chimie Structurale Organique et Biologique, UMR 7613 CNRS, Université Paris 6, 4 place Jussieu, 75252 Paris, France
Received on October 22, 2001; revised on January 7, 2002; accepted on January 7, 2002.
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: 6-N-acetylated-deoxymannojirimycin/affinity chromatography/-L-fucosidase/fucoidan
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In the emerging field of polysaccharide sequencing, a major interest is raised by glycosidases that, because of their enzymatic selectivity, are helpful tools to study sequence-function relationships (Turnbull et al., 1999). Given the algal origin, we have looked for such enzymes in marine environments and have found that the marine mollusk Pecten maximus was a rich source of glycosidases (Daniel et al., 1999
). To our knowledge no fucosidase active on A. nodosum fucoidan has been previously described. We report here the first purification of an
-L-fucosidase from the digestive glands of P. maximus able to release fucose from fucoidan. The physicochemical and catalytic properties of the fucosidase were determined, and its action on fucosylated oligosaccharides and on A. nodosum and F. vesiculosus fucoidans was analyzed by pulsed amperometric detection of the released saccharides.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
|
|
The NMR spectrum of p-nitrophenyl-L-fucose exhibited an anomeric proton H-1-
signal at 5.82 ppm and H-6 signal at 1.16 ppm. When incubated at 25°C with enzyme extract in the NMR tube, 1H NMR spectra revealed a continuous decrease in the signals originating, respectively, from H-1 and H-6 of p-nitrophenyl
-L-fucose. Characteristic signals of free fucose appeared and increased concomitantly. At the beginning of reaction the
anomer was the first to be released, while ß-L-fucose appeared just after and later became the major species because of the mutarotation. After 7 min of reaction, free fucose appeared with an anomeric ratio
/ß of 80/20. After 30 min of reaction, this ratio
/ß was 60/40. This result clearly shows that the hydrolytic mechanism of P. maximus
-L-fucosidase proceeds with retention of configuration.
Kinetic and inhibition studies
Using the synthetic substrate p-nitrophenyl-L-fucopyranoside, we showed that the kinetics of P. maximus fucosidase could be described according the Michaelis-Menten model with a Km value of 650 µM. Several glycosidase inhibitors have been tested (Table III) including imino-sugars, fucose, and fucosamine. The Ki for fucosamine is 316 µM. This value could explain the unsuccessful trials of purification on agarose-
-aminocaproylfucosamine matrix. The reaction product fucose is a better inhibitor than fucosamine, whereas glycosamine derivatives are frequently reported to be better inhibitors (10100 times) than their corresponding carbohydrate (Legler, 1990
). Commercial 1-DMJ is a slightly more potent inhibitor (Ki 34 µM) than 6-N-acetylated-DMJ with a Ki value of 55 µM. As it has been observed with the other purified fucosidases, the most efficient inhibitor is deoxyfuconojirimycin with an inhibition constant Ki of 26 nM, comparable to the Ki value for bovine and human
-L-fucosidases (2.7 and 10 nM, respectively) (Fleet et al., 1985
; Legler et al., 1995
).
|
In addition, when the -L-fucosidase was incubated with several milk oligosaccharides (1 mM) containing fucose at different linkage positions, no other monosaccharide than fucose was detected, even after 1 day of incubation and in spite of the sensitivity of the amperometric detection used. The Fuc
(1
2)Gal linkage in the trisaccharide 2'-fucosyllactose (2'FL) was the most readily hydrolyzed. Among the pentasaccharides assayed, the Fuc
(1
4)GlcNac linkage in lacto-N-fucopentaose (LNFP) II was the most sensitive to the hydrolysis (Table TIV). The Fuc
(1
3)GlcNac linkage in LNFP III and the Fuc
(1
2)Gal linkage in LNFP I were quite insensitive to hydrolysis (around 5%). Regarding these results, no simple relationships can be drawn, but it is likely that the surroundings of the glycosidic bond strongly influences the
-L-fucosidase activity. The high rate of hydrolysis especially concerning 2'FL and LNFP II were obtained with very low amounts of fucosidase (0.025 U), indicating a high activity of the fucosidase toward these fucosylated oligosaccharides.
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The purified enzyme, free from sulfatase and other glycosidases activities, is a tetrameric glycoprotein of 200 kDa. These structural features are shared by most of purified fucosidases in mammals (mouse, rat, human, pig, bovine, monkey) (Johnson and Alhadeff, 1991), mollusks (Charonia lampas, Venus merceneria, Chamelea gallina, Tapes rhomboideus, Littorina littorina, Unio elongatulus) (Cabezas et al., 1983
; Focarelli et al., 1997
) and bacteria (Clostridium perfringens) (Aminoff and Furukawa, 1970
). However, purified enzyme differs in many aspects. Its specific activity of 85 µmol · min1 · mg1 is one of the highest reported, second only to the
-L-fucosidase from Fusarium oxysporum (110 µmol · min1 · mg1) (Yamamoto et al., 1986
). A second interesting characteristic is its stability in relatively acidic medium (pH 4) and high temperatures (above 50°C). Furthermore, in contrast with the other described fucosidases, P. maximus fucosidase is stable at room temperature for several weeks (30% remaining activity after 1 year at room temperature), and it can be freeze-dried without any particular preservative treatment and without significant loss of activity. NMR analysis of the anomeric configuration of the hydrolysis product of the
-L-fucosidase indicates that this enzyme retains the anomeric bond configuration, producing
-anomers that progressively give rise to ß-anomers when mutarotation takes place. The enzyme belongs therefore to the class of "retaining enzymes" (Sinnott, 1990
; Withers, 2001
). Its thermal stability and its high specific activity make the purified
-L-fucosidase a valuable tool in biocatalysis.
Among the different imino-sugars tested, the most potent is 1-deoxyfuconojirimycin (Ki = 26 nM) as for most -L-fucosidases described (Robina et al., 2001
). However, the purified enzyme is about 1000 times less sensitive than many other
-L-fucosidases to 1-DMJ. As an example, the human liver
-L-fucosidase is equally as sensitive toward 1-deoxyfuconojirimycin as toward 1-DMJ (Fleet et al., 1985
). This result suggests a high specificity of the purified enzyme. Except for p-nitrophenyl
-L-fucopyranoside, none of the synthetic glycosides tested (see Materials and methods) were hydrolyzed. In agreement with this specificity, only fucose was released on incubation of
-L-fucosidase with fucosylated oligosaccharides. No other monosaccharide (glucose or galactose) were detected despite the sensitivity of the analytical technique used (PAD).
Though the purified enzyme is specific for -L-fucose, this fucosidase does not exhibit a clear linkage specificity based on the results obtained with fucosylated oligosaccharides. The linkage environment seems to strongly influence the rate of hydrolysis. Indeed, the purified
-L-fucosidase efficiently cleaved the
(1
2) fucose branched in the trisaccharide 2'FL but failed to remove more than 6% of the same fucosyl linkage in the LNFP I. The enzyme is also able to cleave the
(1
4) linkage as observed with the fucosylated pentasaccharide LNFP II but not the
(1
3) linkage of the analog pentasaccharide LNFP III (7.8%). Low linkage specificity has been ascribed for other fucosidases from mollusks, bacteria, and mammals, all of which are active on synthetic substrates like p-nitrophenyl
-L-fucopyranoside or methyl-umbelliferyl
-L-fucopyranoside. Only
-L-fucosidases from almond (Ogata-Arakawa et al., 1977
; Scudder et al., 1990
) and from the microorganisms C. perfringens (Aminoff and Furukawa, 1970
), Xanthomonas manihotis (Wong-Madden and Landry, 1995
), and Streptomyces sp. (Sano et al., 1992
) were reported to have linkage specificity. Further studies using other fucosylated oligosaccharides are being performed to ascertain the specificity of the P. maximus
-L-fucosidase for the
(1
2) and the
(1
4) linkages.
The P. maximus -L-fucosidase catalyzes the hydrolysis of the glycosidic bond of fucosyl unit linked not only to a chromogenic marker or to an oligosaccharide but also to the polysaccharide fucoidan. This result implies the unique property of recognition and hydrolysis of the fucose-fucose linkage because fucoidan is an
-L-fucose-based polysaccharide. To date the molecular structure of algal fucoidan is not fully elucidated. However we and others have recently reported that the structural organization of the A. nodosum fucoidan is based on an
-(1
3) and (1
4)-linked fucose backbone (Daniel et al., 1999
; Chevolot et al., 1999
), unlike to the F. vesiculosus fucoidan, which is based on an
(1
3) fucose backbone (Patankar et al., 1993
). This structural difference in the fucosyl linkage may explain the difference of the
-L-fucosidase activity toward these two fucoidans. The lack of fucose release from the F. vesiculosus fucoidan as well as from the pentasaccharide LNFP III shows the inability of the P. maximus
-L-fucosidase to hydrolyze the
(1
3) fucosyl linkage. Furthermore, the enhancement of the fucose hydrolysis from A. nodosum fucoidan on addition of sulfatase clearly shows that sulfate groups prevent
-L-fucosidase activity. The P. maximus
-L-fucosidase is thus the first enzyme reported to only cleave efficiently the
(1
4)-L-fucose glycosidic bond in a high-molecular-weight polysaccharide. The release of fucose without extensive depolymerization of fucoidan suggests that the hydrolysis of fucose occurs at the nonreducing end of the polysaccharide either at branch or terminal positions.
Enzymes are very useful for the structural analysis of glycans and polysaccharides and for establishing structureactivity relationships in biological systems. The P. maximus -L-fucosidase might be a valuable enzymatic tool to study the structural properties of fucoidan and to improve our knowledge of its biological properties. Recently it has been reported that the fucose branch of the fucoidan could contribute to its biological activities (Patankar et al., 1993
; Pereira et al., 1999
). This
-L-fucosidase is a unique tool to produce fucoidan devoid of such branches (at least the
1
4 branch) and to assess their biological importance. The action of this exo-glycosidase could be completed by other kind of hydrolases (e.g., of the endo type) that could be isolated from P. maximus digestive glands. These additional enzymes, theoretically necessary for fucoidan depolymerization, are currently being sought. Isolation and characterization of such enzymes may enable us to implement a process for the sequential enzymatic hydrolysis of the fucoidan.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Enzyme assays
Glycosidase activity on p-nitrophenyl glycosides was determined in 0.1 M acetate buffer, pH 4, containing 0.2 M NaCl and 3.5 mM synthetic substrates at 60°C for 10 min. The p-nitrophenol released was measured spectrophotometrically at 405 nm ( = 17,919 at pH 9.5) in 100-µl aliquots of the reaction solution diluted in 900 µl of 0.1 M borax buffer, pH 9.5 (John, 1995
). One unit of
-L-fucosidase activity measured on p-nitrophenyl
-L-fucose is defined as the amount of enzyme required to release 1 µmole of p-nitrophenol per min at 60°C and pH 4.0. Fucosidase activity on human milk oligosaccharides was assayed with 2'FL, LNFP I, LNFP II, and LNFP III prepared at 1 mM in 0.1 M acetate buffer, pH 4.0, and incubated at 60°C for 24 h with 0.025 U purified fucosidase. Fucosidase activity on fucoidan was performed by incubating 60 mg of fucoidan with 0.5 U purified fucosidase in 5 ml of 50 mM acetate buffer, pH 5, at 40°C for 5 days. Monosaccharides formed on the enzymatic reaction with the human milk. Oligosaccharides and fucoidan were analyzed by HPLC on an amino column with PAD (see following methods). Samples from the fucoidan reaction were purified by SEC on a PD 10 column (Pharmacia Biotech) eluted with water prior to HPLC analysis to remove high-molecular-weight molecules from samples.
Sulfoesterase activity was routinely assayed with p-nitrocatechol sulfate as substrate using the spectrophotometric determination of p-nitrocatechol at 515 nm (515 = 12,200 M1 cm1) (John, 1995
). The reaction was initiated by the addition of sulfatase in 0.5 ml of 0.1 M acetate buffer, pH 4.5, containing 10 mM substrate. The reaction was carried out at 50°C for 10 min, then 750 µl of 1 M NaOH were added to stop the reaction and to reveal the absorbance at 515 nm. Enzyme specific activity was defined as the amount of µmoles of p-nitrocatechol released per min per mg protein.
Analytical procedures
Hplc analysis of monosaccharides produced during the enzymatic reaction was carried out on an Astec amino column (25 x 0.46 cm) fitted with a guard column (1 x 0.46 cm) (Interchim, France), and with a pulsed amperometric detector Coulochem 2 (Eurosep, France) according to the following pulse potentials: E1 = 0.2 V (700 ms, acquisition delay = 650 ms), E2 = 0.7 V (100 ms), E3 = 0.6 V (100 ms). Elution was performed at the flow rate of 0.8 ml · min1 with 0.1 µm filtered and degassed acetonitrile:water (4:1) mobile phase. A postcolumn delivery system (Dionex, France) added under constant pressure (30 psi) a 0.3 M NaOH solution to the column effluent prior to detection.
High-performance SEC analysis of fucoidan was performed using a Zorbax GF-450 (25 x 0.46 cm) linked to a TSK G2000 SWXL column (30 x 0.78 cm), both equipped with a guard column and connected to a Gilson 132 RI refractometer. The column was eluted with 0.05 M phosphate buffer, 0.15 M NaCl, pH 7.3, at a flow rate of 0.5 ml · min1.
Protein concentration was determined using the Bradford assay system (Biorad) with bovine serum albumin as a standard (Bradford, 1976). Native molecular weight of the
-L-fucosidase was determined by gel filtration chromatography performed on a Superose 12 h column (pharmacia biotech), which was equilibrated and eluted with 0.1 m acetate buffer, ph 5.5, and calibrated with the following molecular weight markers: ferritin (Mr 450,000),
-amylase (Mr 200,000), alcohol dehydrogenase (Mr 150,000), albumin (Mr 67,000), ovalbumin (Mr 43,000), and lactalbumin (Mr 14,200) (pharmacia biotech). Purified
-L-fucosidase was analyzed by 12.5% sdsPAGE after heat denaturation in ß-mercaptoethanol according to the method of Laemmli (1970)
. Protein bands were detected with coomassie blue staining. Glycoprotein staining on polyacrylamide gels was performed with the schiffs reagent (Sigma) after fixation in acetic acid:methanol:water (10:35:55), oxidation by 30 mm periodate in 5% acetic acid (v/v) solution, and neutralization by 10 mm meta-bisulfite in 5% acetic acid (v/v) solution.
Preparation of the 6-amino-DMJ affinity gel
6-N-Amino-DMJ (6-amino-1,5-imino-1,5,6-trideoxy-D-mannitol, as the TFA salt, Figure 1, compound 2) was synthesized from 2-O-allyl-6-[(tert-butyloxycarbonyl)amino]-3,4-di-O-benzyl-1,5-[tert-butyloxycarbonyl) imino]-1,5,6-trideoxy-D-mannitol as previously reported (McCort et al., 2000) with slight modifications. Specifically, the hydroxyl groups were first deprotected by sodium in liquid ammonia followed by deprotection of the amine functions. This compound was coupled to NHS-activated Sepharose 4 Fast Flow (Pharmacia Biotech) following the manufacturer instructions. Briefly, after several washes with 1 mM HCl at 4°C, the medium was equilibrated with the coupling buffer (0.2 M NaHCO3, pH 7.9). Two milliliters of 60 mM 6-amino-DMJ trifluoroacetate were then mixed with 4 ml of medium in the same buffer, and the coupling reaction was performed at 4°C overnight. The unreacted groups were blocked by standing the medium in 0.2 M Tris buffer, pH 8, at room temperature for a few hours. After repeated wash cycles with 0.2 M acetate buffer, pH 4, and 0.2 M Tris buffer, pH 8, the resulting coupled gel was stored in 50 mM acetate buffer, pH 5, containing 20% ethanol (v/v).
Purification of P. maximus -L-fucosidase
The digestive glands from the marine mollusks P. maximus were homogenized with a Warring blender for 10 min in two volumes of extraction buffer (50 mM acetate, pH 5.5, 0.1 M KCl containing 12 µM of phenylmethylsulfonyl fluoride and 10 µM of leupeptin). After 1 h at 4°C under mild agitation, the homogenate was centrifuged (9000 x g) for 20 min. The extract was brought to 30% saturation with (NH4)2SO4, stirred for 45 min, and then centrifuged (10,000 x g) for 25 min. The supernatant was recovered and the concentration of (NH4)2SO4 was gradually increased to 70%. After centrifugation (10,000 x g, 25 min), the recovered precipitate was dissolved in 50 mM acetate buffer, pH 5.5, and dialyzed three times against the same buffer. The dialyzed extract was then washed with 30% (v/v) ethyl acetate to remove lipids. After centrifugation (2,000 x g, 20 min), the aqueous phase was recovered and dialyzed against 25 mM acetate buffer, pH 5.25, prior the first chromatographic step.
Strong cation exchange chromatography.
The dialyzed material was loaded on an SP-Sepharose Fast Flow column (12 x 2.5 cm) (Pharmacia Biotech) which was equilibrated with 25 mM sodium acetate, pH 5.25, at 1 ml · min1. Protein elution was monitored at 280 nm using a 111B UV detector (Gilson). The column was washed with two bed volumes of 25 mM sodium acetate, pH 5.25, then elution was performed with two column volumes of a linear gradient of NaCl (0 to 0.5 M) in the same buffer (flow rate, 1 ml · min1). Fractions containing -L-fucosidase activity measured on p-nitrophenyl
-L-fucose were pooled, diafiltered with 50 mM acetate buffer, pH 7, 0.2 M NaCl and concentrated using an ultrafiltration cell (Amicon) with a 5000-Da membrane.
Immobilized zinc affinity chromatography.
The concentrated enzyme solution was applied at a flow rate of 1 ml min1 to a Chelating Sepharose Fast Flow column (9.5 x 1.6 cm) (Pharmacia Biotech) loaded with Zn2+ and equilibrated with 50 mM acetate buffer, pH 7, 0.2 M NaCl. Protein elution was followed at 280 nm. The column was washed with two bed volumes of the same acetate buffer, then the fucosidase was eluted by 10 mM imidazole in the acetate buffer. Active fractions were pooled, diafiltrated with 50 mM acetate buffer, pH 5, and concentrated to 5 ml using Ultrafree centrifugal filter device 5K (Millipore).
6-Amino-DMJ affinity chromatography.
Routinely, an amount of enzyme extract corresponding to 1 U fucosidase activity was applied onto the 6-amino-DMJ Sepharose 4 Fast Flow column (9.5 x 0.4 cm) equilibrated with 50 mM acetate buffer, pH 5. The column was washed with the same buffer until the absorbance of the eluate (measured at 280 nm) had returned to the baseline. The -L-fucosidase was then eluted by 50 mM fucose in the same acetate buffer. Fucose was removed from the fucosidase solution by diafiltration with Ultrafree centrifugal filter device 5K, and the enzyme was finally concentrated. The
-L-fucosidase was stored at 80°C in acetate buffer 50 mM (pH 5), with no loss of activity observed after several months of storage.
NMR spectroscopy experiments
NMR spectroscopy was performed on a Bruker DMX 500 spectrometer, operating at the proton Larmor frequency of 500.11 MHz. The experiments were performed with a 5-mm probe equipped with self-shielded Z-gradients. Spectra were recorded at 25°C without suppression of HOD signal. Chemical shifts are reported in ppm using sodium 3-trimethylsilylpropanoate as internal reference. One-dimensional spectra were acquired over 32K data points using a spectral width of 5000 Hz.
Mutarotation kinetics of -L-fucose was followed by recording 1H NMR spectra every 2 min for 2 h, after dissolution of 6.1 µmole
-L-fucose in 0.5 ml deuterated 0.1 M acetate buffer, pH 5.5. The ratio between the
- and the ß-anomer was calculated via relative integration of H-1-
/H-1-ß for H-1 signals at 5.20 and 4.55 ppm, respectively, or for H-6-
/H-6-ß methyl signals at 1.21 and 1.25 ppm, respectively. The sample of p-nitrophenyl
-L-fucose was exchanged twice with 99.8% D2O (Sigma) with intermediate lyophilization, and 5.6 µmole was dissolved in 0.5 ml of the previous deuterated acetate buffer. Purified
-L-fucosidase was obtained as a lyophilized powder after a double exchange in D2O and was dissolved in 15 µl of the same deuterated buffer. After recording of the spectrum of the substrate, 15 µl of enzyme solution was added to the NMR tube, which was immediately placed back in the spectrometer. The first spectrum was recorded 2 min after the addition of the enzyme, and then spectra were recorded every 2 min for 4 h.
![]() |
Acknowledgments |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Abbreviations |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Footnotes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Angstwurm, K., Weber, J.R., Segert, A., Bürger, W., Weih, M., Freyer, D., Einhäupl, K.M., and Dirnagl, U. (1995) Fucoidin, a polysaccharide inhibiting leukocyte rolling, attenuates inflammatory responses in experimental pneumococcal meningitis in rats. Neurosci. Lett., 191, 14.[CrossRef][ISI][Medline]
Asano, N., Yasuda, K., Kizu, H., Kato, A., Fan, J.Q., Nash, R.J., Fleet, G.W., and Molyneux, R.J. (2001) Novel -L-fucosidase inhibitors from the bark of Angylocalyx pynaertii (Leguminosae). Eur. J. Biochem., 268, 3541.
Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72, 248254.[CrossRef][ISI][Medline]
Cabezas, J.A., Reglero, A., and Calvo, P. (1983) Glycosidases (fucosidases, galactosidases, glucosidase, hexosaminidases and glucuronidase from some molluscs and vertebrates and neuraminidase from virus). Int. J. Biochem., 15, 243259.[CrossRef][ISI][Medline]
Chevolot, L., Foucault, A., Chaubet, F., Kervarec, N., Sinquin, C., Fisher, A.M., and Boisson-Vidal, C. (1999) Further data on the structure of brown seaweed fucans: relationships with anticoagulant activity. Carbohydr. Res., 319, 154165.[CrossRef][ISI][Medline]
Colliec, S., Boisson-Vidal, C., and Jozefonvicz, J. (1991) A low molecular weight fucoidan fraction from the brown seaweed Pelvetia canaliculata. Phytochemistry, 35, 697700.[CrossRef]
Daniel, R., Berteau, O., Jozefonvicz, J., and Goasdoué, N. (1999) Degradation of algal (Ascophyllum nodosum) fucoidan by an enzymatic activity contained in digestive glands of the marine mollusc Pecten maximus. Carbohydr. Res., 322, 291297.[CrossRef][ISI]
Daniel, R., Berteau, O., Chevolot, L., Varenne, A., Gareil, P., and Goasdoué, N. (2001) Regioselective desulfation of sulfated L-fucopyranoside by a new sulfoesterase from the marine mollusk Pecten maximusapplication to the structural study of algal fucoidan (Ascophyllum nodosum). Eur. J. Biochem., 268, 56175626.
Evans, S.V., Fellows, L.E., Shing, T.K.M., and Fleet, G.W. (1985) Glycosidase inhibition by plant alkaloids which are structural analogues of monosaccharides. Phytochemistry, 24, 19531955.[CrossRef][ISI]
Fleet, G.W., Shaw, A.N., Evans, S.V., and Fellows, L.E. (1985) Synthesis from D-glucose of 1, 5-dideoxy-1, 5-imino-L-fucitol, a potent -L-fucosidase. J. Chem. Soc., Chem. Commun., 13, 841842.
Focarelli, R., Cacace, M.G., Seraglia, R., and Rosati, F. (1997) A nonglycosylated 68-kDa -L-fucosidase is bound to the mollusc bivalve Unio elongatulus sperm plasma membrane and differs from a glycosylated 56-kDa form present in the seminal fluid. Biochem. Biophys. Res. Commun., 234, 5458.[CrossRef][ISI][Medline]
Huang, T.T.R., Ohzu, E., and Yanagimachi, R. (1982) Evidence suggesting that L-fucose is part of a recognition signal for sperm-zona pellucida attachment in mammals. Gamete Res., 5, 355361.[ISI]
John, R.A. (1995) Photometric assays. In Eisenthal, R., and Danson, M.J., eds., Enzyme assay, a practical approach. IRL Press, Oxford, UK, pp. 5992.
Johnson, S.W. and Alhadeff, J.A. (1991) Mammalian -L-fucosidases. Comp. Biochem. Physiol., 99B, 479488.[CrossRef][ISI]
Koshland, D.E. (1953) Stereochemistry and the mechanism of enzymatic reaction. Biol. Rev., 28, 416436.[ISI]
Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680685.[ISI][Medline]
Legler, G. (1990) Glycoside hydrolases: mechanistic information from studies with reversible and irreversible inhibitors. Adv. Carbohydr. Chem. Biochem., 48, 319384.[Medline]
Legler, G., Stutz, A.E., and Immich, H. (1995) Synthesis of 1, 5-dideoxy-1, 5-imino-D-arabinitol (5-nor-L-fuco-1-deoxynojirimycin) and its application for the affinity purification and characterisation of -L-fucosidase. Carbohydr. Res., 272, 1730.[ISI]
Mabeau, S., Kloareg, B., and Joseleau, J.P. (1990) Fractionation and analysis of fucans from brown algae. Phytochemistry, 29, 24412445.[CrossRef][ISI]
McClure, M.O., Moore, J.P., Blanc, D.F., Scotting, P., Cook, G.M.W., Keynes, R.J., Weber, J.N., Davies, D., and Weiss, R.A. (1992) Investigations into the mechanism by which sulfated polysaccharides inhibit HIV infection in vitro. AIDS Res. Human Retroviruses, 8, 1926.[ISI][Medline]
McCort, I., Fort, S., Duréault, A., and Depezay, J.C. (2000) Synthesis and evaluation as glycosidase inhibitors of 2, 5-imino-D-mannitol related derivatives. Bioorg. Med. Chem., 8, 135143.[CrossRef][ISI][Medline]
Mulloy, B., Mourão, P.A.S., and Gray, E. (2000) Structure/function studies of anticoagulant sulfated polysaccharides using NMR. J. Biotechnol., 77, 123135.[CrossRef][ISI][Medline]
Nagumo, T. and Nishino, T. (1996) Fucan sulfates and their anticoagulant activities. In Dumitriu, S., ed., Polysaccharides in medicinal applications. Marcel Dekker, New York, pp. 545574.
Ogata-Arakawa, M., Muramatsu, T., and Kobata, A. (1977) -L-Fucosidases from almond emulsin: characterization of the two enzymes with different specificities. Arch. Biochem. Biophys., 181, 353358.[ISI][Medline]
Patankar, M.S., Oehninger, S., Barnett, T., Williams, R.L.m and Clark, G.F. (1993) A revised structure for fucoidan may explain some of its biological activities. J. Biol. Chem., 268, 2177021776.
Pereira, M.S., Mulloy, B., and Mourão, P.A.S. (1999) Structure and activity of sulfated fucans: comparisons between the regular, repetitive and linear fucans from echinoderms with the more heterogeneous and branched polymers from brown algae. J. Biol. Chem., 274, 76567667.
Preobrazhenskaya, M.E., Berman, A.E., Mikhailov, V.I., Ushakova, N.A., Mazurov, A.V., Semenov, A.V., Usov, A.I., Nifantev, N.E., and Bovin, N.V. (1997) Fucoidan inhibits leukocyte recruitment in a model peritoneal inflammation in rat and blocks interaction of P-selectin with its carbohydrate ligand. Biochem. Mol. Biol. Int., 43, 443451.[ISI][Medline]
Reglero, A. and Cabezas, J.A. (1976) Glycosidases of molluscs: purification and properties of -L-fucosidase from Chamelea gallina L. Eur. J. Biochem., 66, 379387.[Abstract]
Robina, I., Moreno-Vargas, A.J., Fernandez-Bolanos, J.G., Fuentes, J., Demange, R., and Vogel, P. (2001) New leads for selective inhibitors of -L-fucosidases. Bioorg. Med. Chem. Lett., 11, 25552559.[CrossRef][ISI][Medline]
Sano, M., Hayakawa, K., and Kato, I. (1992) Purification and characterization of -L-fucosidase from Streptomyces species. J. Biol. Chem., 267, 15221527.
Scudder, P., Neville, D.C.A., Butters, T.D., Fleet, G.W.J., Dwek, R.A., Rademacher, W.T., and Jacob, G.S. (1990) The isolation by ligand affinity chromatography of a novel form of -L-fucosidase from almond. J. Biol. Chem., 265, 1647216477.
Sinnott, M.L. (1990) Catalytic mechanisms of enzymic glycosyl transfer. Chem. Rev., 90, 11711202.[ISI]
Soeda, S., Ishida, S., Shimeno, H., and Nagamatsu, A. (1994) inhibitory effect of oversulfated fucoidan on invasion through reconstituted basement by murine lewis lung carcinoma. Jpn. J. Cancer Res., 85, 11441150.[ISI][Medline]
Turnbull, J.E., Hopwood, J.J., and Gallagher, J.T. (1999) A strategy for rapid sequencing of heparan sulfate and heparin saccharides. Proc. Natl. Acad. Sci. USA, 96, 26982703.
Winchester, B., Barker, C., Baines, S., Jacob, G.S., Namgoong, S.K., and Fleet, G.W. (1990) Inhibition of -L-fucosidase by derivatives of deoxyfuconojirimycin and deoxymannojirimycin. Biochem. J., 265, 277282.[ISI][Medline]
Withers, S.G. (2001) Mechanisms of glycosyl transferases and hydrolases. Carbohyd. Polym., 44, 325337.[CrossRef][ISI]
Wong-Madden, T. and Landry, D. (1995) Purification and characterization of novel glycosidases from the bacterial genus Xanthomonas. Glycobiology, 5, 1928.[Abstract]
Yamamoto, K., Tsuji, Y., Kumagai, H., and Tochikura, T. (1986) Induction and purification of -L-fucosidase from Fusarium oxysporum. Agric. Biol. Chem., 50, 16891695.[ISI]