From the Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205
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ABSTRACT |
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L-Fucokinase was purified to apparent
homogeneity from pig kidney cytosol. The molecular mass of the enzyme
on a gel filtration column was 440 kDa, whereas on SDS gels a single
protein band of 110 kDa was observed. This 110-kDa protein was labeled
in a concentration-dependent manner by
azido-[32P]ATP, and labeling was inhibited by cold
ATP. The 110-kDa protein was subjected to endo-Lys-C
digestion, and several peptides were sequenced. These showed very
little similarity to other known protein sequences. The enzyme
phosphorylated L-fucose using ATP to form
-L-fucose-1-P. Of many sugars tested, the only other sugar phosphorylated by the purified enzyme was
D-arabinose, at about 10% the rate of
L-fucose. Many of the properties of the enzyme were
determined and are described in this paper. This enzyme is part of a
salvage pathway for reutilization of L-fucose and is also a
valuable biochemical tool to prepare activated L-fucose derivatives for fucosylation reactions.
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INTRODUCTION |
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6-Deoxy-L-galactose
(L-fucose)1 is an
important sugar in animal cells since it is involved in various
recognition reactions of glycoproteins and glycolipids (1). Thus,
oligosaccharides that have -1,2-linked L-fucose are
precursors for blood group A and B antigens (2). In the Lewis blood
group antigens, Gal
1,3 (Fuc
1,4), GlcNAc-R, and
Fuc
1,2Gal
1,3(Fuc
1,4)GlcNAc-R are determinants for
Lewisa and Lewisb blood group antigens. In
addition, fucosylated and sialylated oligosaccharides have been found
to be the recognition molecules for the E- and P-selectins, two members
of the selectin family of cell adhesion molecules (3). These selectins
and their fucosylated (and sialylated) ligands are important in
inflammation and in the recognition of leukocytes for endothelial cells
(4).
The primary pathway for the formation of L-fucose in
procaryotic and eucaryotic cells is from D-mannose via an
internal oxidation reduction and then epimerization of
GDP-D-mannose to produce GDP-L-fucose (5-8).
However, studies in rats showed that radiolabeled L-fucose could be incorporated into glycoproteins (9, 10), suggesting an
alternate route for activation of L-fucose. An
L-fucokinase that synthesizes
-L-fucose-1-phosphate (11) and a
GDP-L-fucose pyrophosphorylase (12) were partially purified
from pig liver. However, the fucokinase preparation had rather broad
substrate specificity with regard to sugar and nucleoside triphosphate, probably because of contaminating enzymes such as hexokinase in the
partially purified fraction. In the present report, we describe the
purification to apparent homogeneity of the pig kidney fucokinase. This
enzyme preparation was very specific for L-fucose, and the only other sugar that could be phosphorylated, at about 10% of the
rate with L-fucose, was D-arabinose. The
fucokinase is also quite specific for ATP as the phosphate donor. This
enzyme should be valuable for the synthesis of large amounts of
L-fucose-1-P, as well as for the formation of radiolabeled
fucose-1-P.
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EXPERIMENTAL PROCEDURES |
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Materials
L-[3H]Fucose (52 Ci/mmol) and other radioactive sugars were purchased from American Radiolabeled Chemicals, Inc., or New England Nuclear Co. L-fucose-1-P, non-radioactive sugars, and nucleoside diphosphate sugars were obtained from Sigma Chemical Co. Various adsorbents were obtained from the following sources: DE-52 from Whatman Chemical Ltd., hydroxylapatite from Bio-Rad, omega-aminohexyl-Agarose and Sephacryl S-300-HR from Sigma Chemical Co. The following materials were obtained from Bio-Rad: Sodium dodecylsulfate (SDS), Acrylamide, Bisacrylamide, Comassie blue, Protein assay reagent. All other chemicals were from reliable chemical sources, and were of the best grade available.
Assay of Fucokinase Activity
Fucokinase activity was assayed by measuring the production of L-fucose-1-P from L-[3H]fucose and ATP. The incubation mixtures contained the following components (final concentrations) in a total volume of 150 µl: 0.1 mM L-fucose, 5 mM ATP, 5 mM MgSO4, 65 mM Tris-HCl buffer, pH 8.0, and various amounts of enzyme at the different stages of purification. Incubations were at 37 °C for 10 min, and reactions were terminated by heating the reaction mixtures in a boiling water bath for 1 min. The incubation mixtures were then applied to columns of DE-52 contained in Pasteur pipettes, and the columns were washed with at least 5 column volumes of 10 mM (NH4)HCO3 to remove the unbound material. The [3H]fucose-1-P was then eluted with 500 mM (NH4)HCO3. Aliquots of the eluates were assayed for their radioactive content by subjecting a portion to scintillation counting.
Purification of the Fucokinase
Preparation of Cytosolic Fraction--
Pig kidneys were obtained
from a local slaughterhouse and were transported to the laboratory on
ice. The fresh kidneys were defatted and cut into pieces that were
washed with cold distilled water. Each kidney piece was homogenized in
a Waring blender in 2 volumes of Buffer A (30 mM Tris-HCl,
pH 7.8, containing 10% glycerol, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, and 1 mM
-mercaptoethanol). The homogenates were centrifuged at 12,000 × g for 30 min in a Beckman J-21 centrifuge. The
supernatant liquid was removed and filtered through six layers of
cheesecloth. The filtered supernatant liquid was then further
centrifuged at 100,000 × g for 45 min. The clarified
supernatant liquid was used as the starting crude extract. All
operations were done at 0-4 °C, unless otherwise specified. In all
cases, fresh kidneys were used as the starting material for
purification since the fucokinase activity was much lower after
freezing the tissue.
DE-52 Column Chromatography-- A 5 × 16 cm column of DE-52 was prepared, and the column was equilibrated with Buffer A. The supernatant liquid from the ultracentrifugation of 400 g of kidney (i.e. about 500 ml of supernatant liquid) was applied to the column, and the column was washed well with Buffer A and then with 800 ml of 0.1 M KCl. The fucokinase was eluted with 1000 ml of a 0.1-0.5 M gradient of KCl. Nine-ml fractions were collected, and every other fraction was assayed for activity and for protein. Active fractions were pooled and brought to 60% saturation by the addition of solid ammonium sulfate. After standing on ice for 15 min, the precipitate was isolated by centrifugation and dissolved in Buffer A containing 1 M ammonium sulfate.
Hydrophobic Chromatography on Macro-Prep Methyl HIC
Support--
The dissolved ammonium sulfate fraction was applied to a
2.5 × 20 cm column of Macro-Prep Methyl HIC Support (Bio-Rad)
that had been equilibrated with Buffer A containing ammonium sulfate. The column was then washed with the equilibration buffer. The kinase
was eluted from the column with a linear gradient of 1-0 M
ammonium sulfate in Buffer A. Active fractions were pooled and concentrated to about 30 ml on an Amicon filtration apparatus using a
PM 30 membrane. The concentrated enzyme was then dialyzed against
Buffer B (30 mM HEPES buffer, pH 7.6, containing 1 mM -mercaptoethanol and 10% glycerol).
Chromatography on Hydroxylapatite-- The dialyzed enzyme was loaded onto a 2.5 × 10 cm column of hydroxylapatite that had been equilibrated with Buffer B. The column was washed with the same buffer and then eluted with a 0-50 mM linear gradient of KH2PO4 in Buffer B. Under these conditions, the kinase bound to the column but not as tightly as other proteins in the preparation. Thus, about 60-70% of the enzyme emerged from the column at 15-25 mM KH2PO4. Some of the enzyme did remain on the column and could be eluted with the bulk of the protein at about 50 mM K2HPO4, in Buffer B.
Gel Filtration Chromatography--
Active fractions, eluted from
the hydroxylapatite column, were pooled and concentrated to about 2 ml
on the Amicon apparatus. The concentrated enzyme preparation was
applied to a 1.5 × 95 cm column of Sephacryl S-300 that had been
equilibrated with Buffer C (25 mM HEPES buffer, pH 7.1, containing 1 mM -mercaptoethanol and 10% glycerol).
Four-ml fractions were collected and assayed for fucokinase activity.
Active fractions were pooled and concentrated to a small volume.
Chromatography on Aminohexyl Agarose--
The concentrated
enzyme fraction from Sephacryl was applied to a 1.5 × 10 cm
column of aminohexyl agarose, which had been equilibrated with Buffer
C. The column was washed with 300 mM NaCl in Buffer C, and
the kinase was eluted with 160 ml of a linear gradient of 300-700
mM NaCl in Buffer C. Fractions containing the active enzyme
were pooled and the NaCl was removed by filtration on an Amicon
apparatus and stored at 80 °C until used for various experiments.
The most purified enzyme preparation gave a single protein band of 110 kDa on SDS gels but was found to still be contaminated with
-mannosidase activity (see "Results"), which also had a
molecular mass of 110 kDa on SDS-PAGE. Thus, fractions from the
aminohexyl-agarose column were assayed for fucokinase and
-mannosidase, and fractions containing fucokinase activity were
incubated with the N3-[32P]ATP probe and
examined by SDS-PAGE and autoradiography to identify the
fucokinase.
Polyacrylamide (Native) Gel Electrophoresis
Preparative polyacrylamide gel electrophoresis was done at 4 °C in tubes containing 7% acrylamide and 10% glycerol as described by Laemmli (13) and using Tris buffer. The pH of the stacking gel was 6.7 and that of the resolving gel was 8.9. The samples of fucokinase were made up to 10% with respect to sucrose and contained bromphenol blue. During electrophoresis, the current was maintained at 3 mA/gel, and the temperature was kept at 4° C. Two samples were run in parallel. One gel was stained with Coomassie Blue to detect proteins while the other gel was cut into 0.25 cm pieces, and the enzyme was eluted by overnight diffusion at 4° C into Buffer A. The various elutions were then assayed for enzymatic activity.
On native gels, the fucokinase (molecular mass of 440 kDa, see
lower band of Fig. 4A) was separated from the
-mannosidase (see upper band of Fig. 4A). To
show that both of these bands were composed of 110-kDa subunits, the
native gel was removed from the tube and laid on its side on top of an
SDS slab gel and polymerized to that gel. Standard proteins were also
combined in a native gel and added to the top of the slab gel. The
proteins in the native gels were then subjected to SDS-PAGE (as seen in Fig. 4B).
Photoaffinity Labeling of the Fucokinase with 8-Azido-[32P]ATP
Enzyme, at various stages of purity, was mixed with 8-azido- ATP[32P] in buffer and allowed to incubate for 20 s in an ice bath. 8-Azido-[32P]ATP was prepared as described previously (14). After incubation, the reaction mixture was exposed to short wave UV light for about 90 s to activate the azido group, and the protein was subjected to SDS-gel electrophoresis to separate the proteins. The gels were dried and exposed to film to locate the radioactive bands and were also stained with Coomassie Blue to locate the various proteins. The specificity of the labeling was determined by examining the effect of various concentrations of unlabeled ATP or other nucleotides on the labeling of the protein by N3-[32P]ATP. Various controls were also run, such as one in which exposure to UV was omitted.
Characterization of the Product
The radioactive sugar phosphate produced in the reaction was isolated by ion exchange chromatography from large scale incubations of [3H]fucose and ATP with purified enzyme. The radiolabeled peak that eluted from DE-52 with a gradient of 0-250 mM (NH4)HCO3 was lyophilized several times to remove the bicarbonate and was then subjected to hydrolysis in various concentrations of HCl to determine the location of the phosphate group. Sugar-1-phosphates are quite sensitive to mild acid hydrolysis (0.05 N), and the phosphate group is lost fairly rapidly, whereas phosphate residues on other hydroxyl groups are quite stable to these conditions. In addition, the product was analyzed by proton NMR to determine the location and anomeric configuration of the phosphate group. Three hundred mHz proton NMR and 31P-decoupled (GARP) NMR on the sample of L-fucose-1-P were performed on a Bruker ARX300 NMR. Data were acquired in D2O at pH 6.0.
Other Methods
Protein was measured by the method of Bradford (15) using bovine
serum albumin as the standard. The molecular weight of the native
fucokinase was determined by gel filtration on Sephacryl S-300 and that
of the subunit by SDS-gel electrophoresis. A number of molecular weight
standards were run including: thyroglobulin (Mr
669,000), apoferritin (Mr 443,000), -amylase
(Mr 200,000), alcohol dehydrogenase
(Mr 150,000), bovine serum albumin
(Mr 66,000), and cytochrome c
(Mr 12,000).
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RESULTS |
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Purification of the Fucokinase-- The pig kidney fucokinase was purified about 5000-fold with a recovery of activity of about 21% using the methods described under "Experimental Procedures." Fig. 1 shows two of the key steps in the purification procedure, i.e. chromatography on hydroxylapatite (panel A) and chromatography on an aminohexyl agarose column (panel B). The hydroxylapatite step gave about a 10-fold purification, whereas chromatography on aminohexyl-agarose gave better than a 6-fold purification. Table I presents a summary of the purification procedure showing the changes in specific activity at each step and the recovery of activity. Based on gel filtration, the native enzyme emerged from the column in the same area as apoferritin and had an estimated molecular mass of about 440 kDa.
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Properties of the Fucokinase-- The purified enzyme was studied to determine its substrate specificity, as well as various other properties of the enzymatic reaction. The enzyme showed a typical pH profile from 5.5 to 8.0 using MES and HEPES buffers, with a sharp pH optimum at about 8.0 in HEPES buffer. However, the pH curve on the alkaline side, between 8.0 and 9.0 in Tris buffer, did not show a sharp optimum. The activity in Tris buffer, pH 8.0, was about 80% of that in HEPES buffer, pH 8.0 (data not shown).
The enzyme had an absolute requirement for a divalent cation for activity. As shown in Fig. 5, Mg2+ gave the best stimulation, with optimum activity being seen at 3 mM concentration. Fe2+ also stimulated the enzyme to nearly the same degree as Mg2+, but in this case, optimum activity occurred at about 10 mM. Co2+ and Mn2+ were also stimulatory, with maximum activity occurring at about 3-5 mM, but the maximum activity was only about one-fourth to one-third of that observed with Mg2+. A variety of other metal ions were tested and found to be inactive, including Ca2+, Cu2+, Fe3+, Hg2+, Mo2+, Ni2+, and Zn2+. However, when Cu2+, Zn2+, and Hg2+ were added at 1 mM concentrations to incubations containing Mg2+, they completely inhibited activity. The specificity of the kinase for various sugar substrates was examined in two different ways. In the first set of experiments, various radiolabeled sugars were tested as phosphate acceptors for the purified enzyme using the ion-exchange chromatography method for assay of activity. Table II shows the results of this experiment. It can be seen that, of all the sugars tested, L-fucose was by far the best substrate and was readily phosphorylated. D-Arabinose, which has the same configuration at carbons 1 through 4 as L-fucose, was also a reasonable substrate for phosphorylation and was about 10% as effective as L-fucose. On the other hand, all of the other sugars were ineffective as phosphate acceptors.
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Tissue Distribution-- To determine whether the fucokinase was present in other tissues besides kidney, crude cytosolic extracts were prepared from various porcine tissues, and each of these extracts was incubated with [3H]fucose, Mg2+, and ATP for various times. The amount of label that bound to DE-52 and the specific activity of each extract are presented in Table V. It can be seen that L-fucokinase activity was present in many different tissues, and in fact, lung and kidney were the tissues with the highest specific activity for this enzyme. Aorta and brain also had reasonably high activity, whereas pancreas, heart, and spleen were the lowest. Crude extracts prepared from cultured MDCK cells and HT-29 cells were also assayed, but no detectable fucokinase activity was found in those extracts.
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Identification of the Product-- The product of the reaction was isolated from large scale incubations of L-fucose with ATP and active enzyme and was purified by ion-exchange chromatography and by paper chromatography. The radioactive fucose product eluted from the DE-52 column in the same position as authentic sugar-1-phosphates, such as glucose-1-P or GlcNAc-1-P. The product was subjected to mild acid hydrolysis in 0.05 N HCl at 100 °C. Aliquots of the hydrolysis mixture were withdrawn at various times after the initiation of heating, and each aliquot was passed through a column of DE-52. The wash and salt elution of the column were subjected to scintillation counting to determine the rate of hydrolysis. The phosphorylated sugar completely lost its charge (and no longer bound to DE-52) within the first 3 min of hydrolysis (data not shown). These data provide convincing evidence that the phosphate group is attached to the anomeric carbon of the sugar.
The sugar-1-P produced in the reaction was subjected to 300 mHz NMR as an aid in the structural characterization as well as to determine the anomeric configuration of the phosphate group. Panel A of Fig. 7 shows 300 mHz NMR proton detection for the anomeric proton region of L-fucose-1-phosphate, and panel B shows the 31P-decoupled spectrum. The J1,2 spin-coupling is 4 Hz for the anomeric proton, consistent with a
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DISCUSSION |
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L-Fucose is an important component of many animal glycolipids and glycoproteins, and turnover of these polymers in the lysosomes must lead to the formation of free L-fucose (9). Thus, it is not surprising to find that certain tissues, especially liver and kidney, contain a specific kinase that can phosphorylate L-fucose to form L-fucose-1-phosphate. In fact, early labeling studies in rats indicated that various tissues are capable of utilizing free L-fucose as a precursor of the L-fucose in glycoproteins (9, 10), suggesting a pathway to reutilize L-fucose.
The presence of the enzyme L-fucokinase was first demonstrated by Ishihara et al. (11) who partially purified this enzyme from pig liver. However, the initial purification of this enzyme was only about 70-fold, and the enzyme preparation still had considerable activity for phosphorylating D-glucose, D-ribose, and L-rhamnose. Thus, that enzyme fraction still probably contained hexokinase, ribokinase, and other enzymatic activities. Interestingly enough, the kinase was also purified about 3500-fold from pig liver by Yurchenko and Atkinson (16). Their enzyme preparation also phosphorylated D-glucose, D-galactose, and D-mannose at about the same rate or better then it phosphorylated L-fucose. These data suggest either that their kinase had a very broad specificity for sugar or that the preparation was still contaminated with hexokinase and galactokinase. Unfortunately, those authors did not examine the nature of the products formed from glucose and mannose to determine whether the phosphate group was in the 1 or 6 position since that would readily have shown whether the reaction was catalyzed by hexokinase. Otherwise, it is difficult to envision how a sugar kinase could catalyze a reaction with four different sugars, all having a different stereochemistry at carbons 2 through 4.
On the other hand, the L-fucokinase described in the present report shows very strong specificity for sugars having the L-galactose configuration at carbons 2 through 4. Thus, only L-fucose and D-arabinose were active as phosphate acceptors although D-arabinose was only about 10% as effective as L-fucose. In addition, the enzyme only utilized ATP as the phosphate donor in contrast to the enzyme reported by Ishihara et al. (11), which could also use CTP, UTP, and GTP as phosphate donors. Thus, it is not clear whether the enzyme reported here is a new enzyme or whether it is the same protein as described earlier but has been purified much more extensively to apparent homogeneity. We sent the purified enzyme to Harvard Microsystems for amino acid sequencing and obtained the sequences of three peptides. These sequences do not show any more then 40% homology to known sequences by the BLAST search. Thus, our protein is clearly not very closely related to other reported kinases.
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ACKNOWLEDGEMENTS |
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We are grateful to the Louisiana State University NMR facility for the analysis of the phosphorylated sugar and especially to Dr. Roger Laine for the interpretation of the data and for helpful and stimulating discussions pertaining to these studies.
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FOOTNOTES |
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* This study was supported by a grant from the National Institutes of Health (HL-17783) and by funds from Cytel Co.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Contributed equally to this work and should be considered senior
co-author.
§ To whom correspondence should be addressed. Fax: 501-686-8169.
1 The abbreviations used are: L-fucose, 6-deoxy-L-galactose; PAGE, polyacrylamide gel electrophoresis; MES, 4-morpholineethanesulfonic acid.
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REFERENCES |
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