Báo cáo khóa học: Cloning and expression of murine enzymes involved in the salvage pathway of GDP-L-fucose ppt

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Báo cáo khóa học: Cloning and expression of murine enzymes involved in the salvage pathway of GDP-L-fucose ppt

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Cloning and expression of murine enzymes involved in the salvage pathway of GDP- L -fucose L -fucokinase and GDP- L -fucose pyrophosphorylase Jaana Niittyma¨ki 1 , Pirkko Mattila 2 , Christophe Roos 2 , Laura Huopaniemi 1 , Solveig Sjo¨ blom 1 * and Risto Renkonen 1,3 1 Department of Bacteriology and Immunology, Haartman Institute and Biomedicum, University of Helsinki; 2 MediCel, Helsinki; 3 HUCH Laboratory Diagnostics, Helsinki University Central Hospital, Finland In the salvage pathway of GDP- L -fucose, free cytosolic fucose is phosphorylated by L -fucokinase to form L -fu- cose-1-phosphate, which is then further converted to GDP- L -fucose in the reaction catalyzed by GDP- L -fucose pyrophosphorylase. We report here the cloning and expression of murine L -fucokinase and GDP- L -fucose pyrophosphorylase. Murine L -fucokinase is expressed as two transcripts of 3057 and 3270 base pairs, encoding proteins of 1019 and 1090 amino acids with predicted molecular masses of 111 kDa and 120 kDa respectively. Only the longer splice variant of L -fucokinase was enzy- matically active when expressed in COS-7 cells. Murine GDP- L -fucose pyrophosphorylase has an open reading frame of 1773 base pairs encoding a protein of 591 amino acids with a predicted molecular mass of 65.5 kDa. GDP- L -fucose, the reaction product of GDP- L -pyrophosphory- lase, was identified by HPLC and MALDI-TOF MS analysis. The tissue distribution of murine L -fucokinase and GDP- L -fucose pyrophosphorylase was investigated by quantitative real time PCR, which revealed high expres- sion of L -fucokinase and GDP- L -fucose pyrophosphory- lase in various tissues. The wide expression of both enzymes can also be observed from the large amount of data collected from a number of expressed sequence tag libraries, which indicate that not only the de novo pathway alone, but also the salvage pathway, could have a signi- ficant role in the synthesis of GDP- L -fucose in the cytosol. Keywords: GDP- L -fucose; L -fucokinase; GDP- L -fucose pyrophosphorylase; salvage pathway; molecular cloning. L -Fucose is an important monosaccharide in the complex carbohydrates of mammals. It decorates N- and O-linked glycoproteins and glycolipids [1] or is covalently linked to some serine or threonine residues of proteins [2]. Various functions have been established in biological processes for fucose residues that are present in the terminal chains of oligosaccharides of membrane bound or secreted molecules [3]. Fucosylated glycans form ABO and Lewis blood group antigens in humans [4,5]. Glycans that contain a(1,3)- fucosylated modifications, e.g. sialyl Lewis x-type glycans, have an important role in inflammation. They initiate extravasation of leukocytes by mediating their tethering and rolling on the endothelium by decorating the leukocyte and endothelial cell counter receptors for selectin family of cell adhesion molecules [6,7]. Fucosylation also seems to play an important role in fertilization [8,9], development [10–13], tumor metastasis [14] and programmed cell death [15]. Fucosylation requires GDP- L -fucose as a donor of fucose and as a substrate for fucosyltransferases. Two different cytosolic pathways lead to formation of GDP- L -fucose. The constitutively active de novo pathway involves conversion of GDP-a- D -mannose to GDP-b- L -fucose by two enzymes, GDP- D -mannose-4,6-dehydratase (GMD) and GDP-4- keto-6-deoxy- D -mannose-3,5-epimerase-4-reductase (FX) [16,17]. In the alternative biosynthetic pathway, i.e. the ÔsalvageÕ metabolism, L -fucokinase synthesizes L -fucose-1- phosphate from L -fucose and ATP. GDP- L -fucose pyrophosphorylase further catalyzes the formation of GDP- L -fucose from L -fucose-1-phosphate and GTP. The salvage pathway utilizes fucose obtained from extracellular sources or from intracellular degradation of glycoproteins and glycolipids [Fig. 1]. Correspondence to: R. Renkonen, Department of Bacteriology and Immunology, Haartman Institute and Biomedicum, PO Box 63, FIN-00014 University of Helsinki, Helsinki, Finland. Fax: + 358 9 1912 5155, Tel: + 358 9 1912 5111, E-mail: Risto.Renkonen@Helsinki.Fi Abbreviations: CDS, coding sequence; EST, expressed sequence tag; FX, GDP-4-keto-6-deoxy- D -mannose-3,5-epimerase-4-reductase; GMD, GDP- D -mannose-4,6-dehydratase; LADII, leukocyte adhesion deficiency type 2. Enzymes: GDP-mannose 4,6-dehydratase (EC 4.2.1.47); GDP-4- keto-6-deoxy- D -mannose 3,5-epimerase/4-reductase (EC 1.1.1.187); L -fucokinase (EC 2.7.1.52); GDP- L -fucose pyrophosphorylase (EC 2.7.7.30). Note: Nucleotide sequence data are available in the DDBJ/EMBL/ GenBank databases under the accession numbers AJ297482, AJ534942 and AJ276067. *Present address: Department of Biosciences, Division and Genetics, University of Helsinki, Finland. (Received 6 October 2003, accepted 30 October 2003) Eur. J. Biochem. 271, 78–86 (2004) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03904.x L -fucokinase and GDP- L -fucose pyrophosphorylase were first discovered in pig liver [18,19]. To date L -fucokinase has been partially purified and characterized from porcine liver [19] and thyroid gland [20], and purified to apparent homogeneity from pig kidney [21]. Furthermore, the gene encoding human fucokinase has been identified [22]. GDP- L -fucose pyrophosphorylase has been purified from porcine kidney and the corresponding gene has been cloned from human [23]. In the present study we have cloned the murine genes coding for the enzymes involved in the salvage pathway of GDP- L -fucose. L -fucokinase and GDP- L -fucose pyro- phosphorylase were expressed in COS-7 cells, and the enzyme activities were determined. Experimental procedures Cloning of mouse L -fucokinase Based on the sequence from three published pig fuco- kinase peptides [21], a portion of the mouse fucokinase sequence was resolved through tBlastn and FastA- searches of the EMBL/GenBank/DDBJ sequence data- base [24,25]. Using expressed sequence tags (ESTs), primers corresponding to putative fucokinase sequence were designed. A region of the mouse fucokinase sequence was amplified by PCR from the cDNA of mouse kidney (QuickClone cDNA, Clontech, Palo Alto, CA, USA), cloned into blunt II-TOPO vector (Invitro- gen, Carlsbad, CA, USA) and sequenced. This sequence was used as a query tool for further sequence database searches and sequence alignments. The IMAGE clone 4190449 (accession number BF538673) was obtained from MRC geneservice (Cambridge, UK) and sequenced. This clone, which was identified as containing a putative fucokinase, contained the full coding sequence (CDS) of L -fucokinase. RT-PCR was performed to confirm the relevance of the IMAGE clone sequence. Mouse kidney total RNA (Ambion, Inc., Austin, TX, USA) was used as a template in the first strand cDNA synthesis (Superscript First Strand synthesis system for RT-PCR, Invitrogen). Primers for RT-PCR were designed accord- ing to the sequence data gained from IMAGE clone 4190449. The gene-specific primer for in vitro reverse transcription, was 5¢-TAGCAGCAGACTTGAAGAGG TA-3¢. PCR was performed by using the forward primer 5¢-GCCAGAATGGAGCAGTCAGAGGGAGTC-3¢ and the reverse primer 5¢-GCAGCTCTAGGTGGTGCCCA CTTCAGAG-3¢. The PCR products were cloned into pCRÒ-XL-TOPOÒ vector (Invitrogen) and sequenced. Two clones were identified displaying two putative splice variants [Fig. 2]. Expression of fucokinase cDNAs The two splice variants of fucokinase were subcloned into the XbaI site of a pQM vector containing a C-terminal E2-Tag/A (Quattromed Ltd, Tarto, Estonia). The forward primer was 5¢-ATCTCTAGAATGGAGCAGTCAGA G-3¢ and the reverse primer was 5¢-ATCTCTAGAGGT GGTGCCCACTTC-3¢. All primers contained the XbaI restriction enzyme recognition site (underlined in the oligonucleotide sequences). Long and short splice variants of fucokinase were transiently transfected into COS-7 cells by lipofectamine 2000 (Invitrogen) according to the manu- facturer’s instructions. After 48 h, the transfected cells were lysed in 50 lLof50m M Tris/HCl (pH 7.8), 150 m M NaCl, 1% (v/v) Triton X-100, and incubated on ice for 1 h with a protease inhibitor cocktail (BD, Erembodegem, Belgium). Protein concentrations were determined using bicinchoninic acid protein reagent (Pierce Chemical Co., Rockford, IL, USA). Fig. 1. Synthesis of GDP- L -fucose in mammals. The constitutively active de novo pathway converts GDP- D -mannose into GDP- L -fucose via oxidation, epimerization and reduction catalyzed by two enzymes, GMD and FX. In the alternative salvage pathway, free fucose is delivered to cytosol from extracellular sources (shown) or from lyso- somal degradation of glycoconjugates (not shown). L -fucose is phos- phorylated by L -fucokinase to form L -fucose-1-phosphate, which is converted to GDP- L -fucose in the reaction catalyzed by GDP- L -fucose pyrophosphorylase. GDP- L -fucose is then transported into the Golgi. Fig. 2. Gene structures of the short and the long splice variants of mouse fucokinase (A) and human fucokinase (B). The long splice variant of mouse fucokinase contains exons 1–20, 21a, 22, 23a and 24. The short splice variant contains exons 1–20, 21b, 23b and 24. The human fucokinase has a similar gene structure to the long splice variant of mouse fucokinase at the 3¢ end. Ó FEBS 2003 The salvage pathway of GDP- L -fucose in mouse (Eur. J. Biochem. 271)79 COS-7 cell lysates (30 lg) were detected by Western blotting using anti-(E2-Tag) primary mAb (Quattromed) and anti-(mouse IgG) HRP-conjugated secondary anti- body. Detection was performed using enhanced chemilu- minescence (Amersham Biosciences, Bucks, UK) according to standard methods. Fucokinase activity assay Cell lysate (100 lg) was assayed in a 100 lL reaction mixture containing 50 m M Tris/HCl (pH 8.0), 5 m M MgSO 4 , 150 000 c.p.m. L -[ 3 H]fucose (specific activity 63.0 CiÆmmol )1 , Amersham), 0.1 m M fucose (Sigma, St Louis, MO, USA), 5 m M ATP and 5 m M NaF (Sigma) in final concentrations. The reaction mixture was incubated at 37 °C for 30 min and terminated with 100 lL of ethanol. The incubation mixture was applied to two 10 cm DEA- Bond Elut column (Varian, Palo Alto, CA, USA), which was then washed with four column volumes of 10 m M NH 4 HCO 3 to remove the unbound material. The [ 3 H]fu- cose-1-P was eluted with 2 mL of 250 m M NH 4 HCO 3 .The eluate (400 lL) was counted with liquid scintillation and luminescence counter (Wallac Trilux, Turku, Finland). Cloning of GDP- L -fucose pyrophosphorylase The 3¢ end of the pyrophosphorylase gene was cloned from the mouse kidney UNI-ZAP XR lambda cDNA library (Stratagene, La Jolla, CA, USA) by screening of approxi- mately 1 · 10 6 recombinant plasmids. The published human GDP- L -fucose pyrophosphorylase (accession num- ber AF017445) [23] was used in a BLAST search to locate mouse ESTs corresponding to the putative pyrophospho- rylase. According to the EST sequence (AA422658), the forward primer 5¢-GAGTATTCTAGATTGGGGCCT GA-3¢ and reverse primer 5¢-TGTGGACTGCACGCA TTTTCC-3¢ were designed. PCR was performed using mouse liver cDNA (QuickClone cDNA, Clontech) as a template. The 330 bp PCR product was labelled with [ 32 P]dCTP[aP] using the Multiprime DNA labelling kit (Amersham Biosciences, Buckinghamshire, UK) according to the manufacturer’s protocol. The labelled probe was used in colony hybridization according to standard methods. Theentire5¢ end was resolved by 5¢ RACE-PCR (Robust RT-PCR kit, Finnzymes, Espoo, Finland) using mouse kidney mRNA (Clontech) as a template. PCR was performed using the 5¢ RACE synthesis primer AP1 (Clontech), 5¢-CCATCCTAATACGACTCACTATAGG GC-3¢ and the gene-specific reverse primer, 5¢-GACTCC AGGCCTCATGTTTGAGGGGAAATCCACGTAC-3¢. The second round PCR was performed with a nested adaptor primer AP2 (Clontech), 5¢-ACTCACTATAGG GCTCGAGCGGC-3¢ together with a nested gene-specific primer, 5¢-CAAACACTCAAGGGAACAAAG-3¢.All PCR products were cloned into pCRÒ-Blunt II-TOPOÒ vector (Invitrogen). The enzymatic activity of GDP-fucose pyrophosphorylase The coding sequence of pyrophosphorylase was amplified by PCR using the forward primer, 5¢-AAT GGTACC ATGGCGTCTCTCCGCGA-3¢ and the reverse pri- mer, 5¢-CAC GGATCCTTAAGATTTCTCTAAATCAG-3¢ creating KpnI and BamHI restriction enzyme recognition sites (underlined), respectively. Subcloning of the PCR product into a pCDNA3.1(+) vector (Invitrogen) and the transient transfection into COS-7 cells were per- formed as above. The cells were lysed on ice with 50 m M Tris/HCl (pH 7.5) including protease inhibitor cocktail (Pharmingen), with sonication for 3 · 15 s (Branson Sonifier 450, Heinemann, Schwa ¨ bich Gmund, Austria). Cell lysates (60 lg) were incubated in a 50 lL reaction mixture containing 0.5 M Tris/HCl (pH 7.8), 200 m M MgCl 2 ,10m M b- L -Fuc-1P (Sigma), 100 m M GTP, 0.5 U inorganic pyrophosphorylase (Sigma) at 37 °C for 30 min. Nucleotide sugars were purified from the cell lysates as described by Rabina et al. [26] and analyzed by ion-pair reversed-phase HPLC on a Discovery HS C18 column (0.46 · 25 cm; Supelco Inc., Pennsylvania, PE, USA) at a flow rate of 1 mLÆmin )1 . A linear gradient of 0–1.5% (v/v) acetonitrile in 20 m M triethyammonium acetate buffer (pH 7.0) over 35 min was used and the effluent was monitored at 254 nm. The amount of synthesized GDP- L -fucose was calculated using the peak areas of external nucleotide sugar standards GDP- D -mannose, GDP- D -rhamnose [27] and GDP- L -fucose (Calbiochem, San Diego, CA, USA). The fraction containing the putative GDP- L -fucose was collected from the HPLC assay for further analysis with MALDI-TOF MS. MALDI-TOF-MS MALDI-TOF MS was performed with a Biflex III mass spectrometer (Bruker Daltonics, Bremen, Germany). Nuc- leotide sugars were investigated in a 2,4,6-trihydroxy- acetonephenone–acetonitrile–aqueous ammonium citrate matrix as described previously [26], utilizing the reflector negative-ion mode with delayed extraction. External calib- ration was performed with TDP- D -rhamnose (a generous gift from P. Messner, Universita ¨ tfu ¨ r Bodenkultur Wien, Wien, Austria) and UDP-GlcNAc (Sigma). GDP- L -fucose (Calbiochem) was used as a positive control. Reverse transcription and quantitative real time PCR Fucokinase and pyrophosphorylase mRNA expression in different tissues was detected by quantitative real time PCR. Ambion’s Mouse Total RNA (kidney, liver, brain, ovary, testicle, heart, lung, spleen) was used for the first strand cDNA synthesis. For each tissue, 1 lg of total RNA was reverse transcribed with random hexamers using the Invi- trogen SuperScriptÒ cDNA synthesis kit according to the manufacturer’s instructions. Parallel reactions in the absence of SuperScript IIÒ (–RT controls) were performed to assess the degree of contaminating genomic DNA. The resulting cDNA samples were subjected to real time quantitative PCR assay [28] to detect the expression levels of pyrophosphory- lase and long and short splice variants of fucokinase. Primers and probes were designed using the PRIMER EXPRESS program (Version 1, PE Applied Biosystems, Foster City, CA, USA), a software tool provided with the ABI 7000 Sequence Detection System (PE Applied Biosystems). Forward and reverse primers were positioned as close as possible to each 80 J. Niittyma ¨ ki et al. (Eur. J. Biochem. 271) Ó FEBS 2003 other without overlapping the probe. Probes were synthes- ized incorporating the fluorescent reporter FAM (6-carboxy- fluorescein) at the 5¢ end and the quencher TAMRA (6-carboxy-tetramethyl-rhodamine) at the 3¢ end. One microlitre of freshly synthesized cDNA was ampli- fied in a total volume of 25 lL containing 1· Universal Master Mix (PE Applied Biosystems) on an ABI Prism 7000 Sequence Detection System. Assays for each transcript were carried out as duplicates on the same plate and real time PCR amplification was repeated twice. Any inefficiencies in RNA input or reverse transcription were corrected by normalization to a housekeeping gene (18S rRNA Control Reagents, PE Applied Biosystems). Primer concentrations used were 900 n M /300 n M (forward/reverse) for the long splice variant of fucokinase, 900 n M /900 n M (forward/ reverse) for the short splice variant of fucokinase, and 300 n M /900 n M (forward/reverse) for pyrophosphorylase. The concentration of the double labelled probe was 200 n M for the long variant of fucokinase and pyrophosphorylase, and 300 n M for the short fucokinase splice variant. Relative amounts of the three mRNAs analyzed were based on standard curves (Applied Biosystems User Bulletin 2) prepared by a serial dilution of control cDNA. Results Cloning of putative mouse L -fucokinase and sequence analysis Using the three known pig fucokinase peptides [21] as probes, part of the putative murine fucokinase sequence was identified from mouse genomic sequence from the EMBL/ GenBank/DDBJ database. This sequence was cloned from mouse kidney cDNA and used as a query in order to find thecompletesequencefromthedatabase.TheIMAGE clone 4190449 contained the full CDS of a putative mouse L -fucokinase, which was utilized in the design of primers for RT-PCR. Two putative cDNAs of different sizes were cloned representing two splice variants of L -fucokinase. The long splice variant of L -fucokinase consisted of 3270 bp, encoding a protein of 1090 amino acids. The sequence of the shorter cDNA was similar to the sequence of the IMAGE clone 4190449, consisting of 3057 bp. The short splice variant did not code for amino acids 921–992 present in the long splice variant, thus the short version consisted of 1019 amino acids [Fig. 3]. The long splice variant of L -fucokinase contains exons 1–20, 21a, 22, 23a and 24 whereas the short one contains exons 1–20, 21b, 23b and 24. As can be seen in Fig. 2, exons 21b and 23b are wholly included in the longer variants of these exons (21a and 23a respectively). The splice junction from exon 20 to exon 21a or 21b is not affected, neither is the splice junction between exon 23a or 23b, and exon 24. In conclusion, the alternative splicing maintains the reading frame along the entire protein, therefore the protein variants are identical in the amino-terminal end up to the alternative splice area, in addition to the carboxy-terminal end after the alternative splice area. There are three methionine codons (ATG) within a 300 bp region at the upstream end of the longest open reading frame in the mouse fucokinase mRNA sequence (accession number AJ534942). The first ATG is estimated to be the most probable CDS initiation site based on a probabilistic model using multiple parameters, including the Kozak translation initiation signal, as implemented in the GENSCAN analysis tool [28]. Expression of fucokinase in mammalian cells The two splice variants of the murine fucokinase genes were expressed in COS-7 cells in frame with a 10-amino acid E2- Tag present in the pQM vector. The molecular masses of fucokinase proteins were determined by Western blot analysis; the tagged long splice variant had a mass of 125 kDa and the tagged short splice variant a mass of 115 kDa. Both E2-Tagged splice variants had slightly greater molecular masses than the predicted 120 and 111 kDa proteins, respectively [Fig. 4]. The production of L -fucose-1-phosphate from L -[ 3 H]fu- cose and ATP was measured in order to determine whether the expressed splice variants of fucokinase were functionally active. The long splice variant showed significant enzyme activity; the specific enzyme activity was determined to be 598.5 pmolÆmg )1 Æh )1 in transfected COS-7 cells. The activity of the short splice variant was only marginally higher (13.7 pmolÆmg )1 Æh )1 ) than the background in the COS-7 cells (11.4 pmolÆmg )1 Æh )1 ). Human L -fucokinase, IMAGE clone 4179554 (AJ441184) [22], was also transfected into COS-7 cells and assayed in regard to fucokinase activity. The specific enzyme activity of the human L -fucokinase was the same level, 12.3 pmolÆmg )1 Æh )1 , as the activity of the shorter mouse splice variant and the vector control (Fig. 5A). L -fucokinase activity is present in many different tissues, and exhibits high activity in kidney [21]. The COS-7 cell line is derived from monkey kidney cells and thus has some intrinsic fucokinase activity. In order to discriminate the possible fucokinase activity of a short splice variant from the kidney cell backround, the short splice variant was also transfected into epithelial HeLa S3-cells. The relatively weak enzymatic activity of the short splice variant could be detected in HeLa cells; the specific enzyme activity was 30.6 pmolÆmg )1 Æh )1 whereas the specific activity of the mock control was 8.4 pmolÆmg )1 Æh )1 (Fig. 5b). Cloning of murine GDP- L -fucose pyrophosphorylase The cloned human pyrophosphorylase (accession number AF017445) [23] was used as a query in BLAST searches to find a mouse EST corresponding to the putative pyro- phosphorylase. Using this mouse EST as a probe, the 3¢ end of the GDP- L -fucose pyrophosphorylase was cloned from a mouse kidney cDNA library by screening  1 · 10 6 recom- binant plasmids. The 5¢ end of the sequence was resolved by the RACE-PCR method, using mouse kidney mRNA as the template as described in Experimental procedures. The isolated cDNA consisted of 3480 bp, and the predicted CDS encoded a protein of 591 amino acids [Fig. 6]. Pyrophosphorylase activity assay and the identification of GDP- L -fucose Because we could detect only a faint protein band in SDS/ PAGE from cell lysate with the estimated molecular mass of 65.5 kDa that relates to GDP- L -fucose pyrophosphorylase Ó FEBS 2003 The salvage pathway of GDP- L -fucose in mouse (Eur. J. Biochem. 271)81 (data not shown), we decided to identify accurately the product of a GDP- L -pyrophosphorylase assay. The cell lysate expressing the pyrophosphorylase gene was incubated with L -fucose-1-phosphate and GTP, and the resulting product of the reaction was analyzed by ion-pair reversed- phase HPLC. The analysis revealed a peak with the same retention time as the GDP- L -fucose standard (F) at 29.6 min in a sample containing the pyrophosphorylase, whereas the vector control gave only a faint peak at 29.7 min [Fig. 7]. The peak was purified and subjected to further analysis by MALDI-TOF MS, which gave a single peak at 588.08 m/z, thus being identical to the GDP- L - fucose control. Quantitative PCR and tissue distribution levels of L -fucokinase and pyrophosphorylase The primer and probe sequences and their positions in the mRNA sequence, for GDP- L -fucose pyrophosphorylase and the short and long splice variants of L -fucokinase, are listed in Table 1. Various mouse tissues were analyzed for the expression of the three enzymes (GDP- L -fucose pyrophosphorylase, and short and long splice variants of L -fucokinase) involved in the salvage pathway of GDP- L -fucose, to elucidate the possible differences between the various tissues. Moreover, the ratio of long to short splice variants of L -fucokinase in Fig. 3. Nucleotide sequence and deduced amino acid sequence of mouse L -fucokinase. The predicted amino acid sequence for the coding area of the long splice variant of fucokinase consists of 1090 amino acids. Due to alternative splicing, the amino acids 921–992 (bold letters) are not coded in the short splice variant of fucokinase. The amino acids corresponding to the published peptide sequences of pig fucokinase [21] are underlined. The sequence data of the short splice variant is available in the EMBL/GenBank/DDBJ Nucleotide Sequence Databases under Accession No. AJ297482 and the long splice variant under the Accession No. AJ534942. 82 J. Niittyma ¨ ki et al. (Eur. J. Biochem. 271) Ó FEBS 2003 different tissues was also determined (Table 2). Relative expression levels, shown in Fig. 8, were calculated following normalization to 18S RNA. In the subsequent calculations, expression levels of those enzymes found in mouse liver were assigned a relative expression value of one. The expression of both splice variants of L -fucokinase was found to be relatively high in brain, ovary, testis and kidney. In spleen, heart and lung the expression was lower. When calculating the ratio between the long and short splice variants of fucokinase it could be seen that the long splice variant was more abundantly expressed in liver, kidney, ovary, testis, spleen and heart. In the lung the expression levels were equal, whereas in brain the expression of the short splice Fig. 5. Fucokinase activities of the cell lysates of COS-7 cells (A) and HeLa cells (B) transfected with the fucokinase cDNAs. Enzyme activity is expressed as pmol of L -[ 3 H]fucose incorporated onto ATP per hour devided by the total protein content. (A) Enzyme activities of COS-7 cells, transfected with the short and long splice variants of mouse fucokinase (mFK) and human fucokinase (hFK, AJ441184). (B) Fucokinase activities of HeLa cells transfected with vector or the short splice variant of mouse fucokinase. Fig. 4. Western blot analysis of the expressed murine L -fucokinase in COS-7 cells detected with E2-Tag antibodies. Lane 1, negative COS-7 cell control; lane 2, short splice variant of mouse fucokinase and lane 3, long splice variant of mouse fucokinase. Fig. 6. Nucleotide sequence and deduced amino acid sequences of murine GDP- L -fucose pyrophosphorylase. The 3.5 kb nucleotide sequence predicts an amino acid sequence of 590 residues for the coding region of GDP- L -fucose pyrophosphorylase. The sequence data is available in the EMBL/GenBank/DDBJ Nucleotide Sequence Databases under Accession No. AJ276067. Ó FEBS 2003 The salvage pathway of GDP- L -fucose in mouse (Eur. J. Biochem. 271)83 variant was higher than that of the long L -fucokinase splice variant. The expression pattern of GDP- L -fucose pyro- phosphorylase resembles the pattern of L -fucokinase, i.e. expression was high in brain, ovary, testis and kidney. Again, the expression levels were lower in liver, spleen, heart and lung (Fig. 8C). Discussion The de novo synthesis of GDP- L -fucose, that converts GDP- D -mannose to GDP- L -fucose, is evolutionary conserved and the enzymes involved in this pathway have been cloned from several bacteria [17], plants [29] and mammals [30]. In addition, the de novo synthesis of GDP- L -fucose has been characterized in silico from the fruit fly [31]. The alternative pathway of GDP- L -fucose synthesis, the salvage pathway, allows cells to activate monosaccharides that come from nutrition or from lysosomal degradation of glycoproteins and glycolipids. The sugars are phosphorylated by kinases and activated by pyrophosphorylases. To date the salvage pathway of GDP- L -fucose has been identified only in mammals [21,23]. The specific salvage pathway is also found for UDP-galactose, UDP-glucuronic acid and UDP-N- acetylgalactosamine [32]. The salvage pathway of GDP- L -fucose involves L -fuco- kinase which catalyzes the transfer of phosphate from ATP to free L -fucose, forming L -fucose-1-phosphate. GDP- L - fucose pyrophophorylase then condensates L -fucose- 1-phosphate with GTP to form GDP- L -fucose. In the present study we have cloned the murine enzymes involved in the salvage pathway of GDP- L -fucose and expressed them as functionally active enzymes. Two splice variants of L -fucokinase were cloned, but only the long splice variant was enzymatically active when expressed in mammalian cells. The short splice variant did not show significant Table 1. Probe and primer sequences in quantitative PCR. FK short, short splice variant of L -fucokinase; FK long, long splice variant of L -fucokinase; PP, GDP-l-fucose pyrophosphorylase; F, forward primer; R, reverse primer; P, probe. Target gene Primer/Probe sequence Starting position in mRNA Length of amplicon FK short F 5¢-CTGAGGGTTTGTCCCAGAA-3¢ 2786 103 bp R5¢-GGCTTTGGCCATACGCATAC-3¢ 3081 P a 5¢-ACGGCCAGCGGCTCGCA-3¢ 3037 FK long F 5¢-GCAGGACGTGCTGAGGAACT-3¢ 2865 64 bp R5¢-CAGTCTGCGGGCATTCTGT-3¢ 2911 P a 5¢-CCACAACGGGCAACCGAGCG-3¢ 2889 PP F 5¢-AGCTGGGCTTACAATCCATAGCT-3¢ 1118 79 bp R5¢-TGAATGACACAGGCTGTTCCA-3¢ 1176 P5¢-AGTGTCTCTCCAAGTGTTCCTGAGCGCT-3¢ 1144 a Probe is antisense strand. Table 2. Ratio of long splice variant to short splice variant of L -fucokinase. Tissue Ratio Liver 1.52 Kidney 1.34 Ovary 1.40 Testis 1.28 Spleen 3.21 Brain 0.77 Heart 1.15 Lung 0.99 Fig. 7. Ion-pair reversed-phase HPLC analysis of the product of the enzymatic reaction catalyzed by GDP- L -fucose pyrophosphorylase. (A) Vector control in COS-7 cell lysate; (B) putative mouse pyrophos- phorylase in COS-7 cell lysate; (C) GDP-sugar standards, 500 pmol of each. M, GDP- D -mannose, 18.6 min; R, GDP- D -rhamnose, 24.4 min; F, GDP- L -fucose, 29.6 min. 84 J. Niittyma ¨ ki et al. (Eur. J. Biochem. 271) Ó FEBS 2003 enzymatic activity, but was expressed abundantly in many tissues, especially in brain, which may indicate an uniden- tified role for this variant. When comparing both splice variants of mouse L -fuco- kinase cDNA sequence with the previously published human L -fucokinase cDNA sequence (accession number AJ441184) [22], it can be observed that the human fucokinase cDNA is similar to the long splice variant of mouse fucokinase cDNA at the 3¢ splice region. The first and third methionines in the murine sequence, in the upstream end of the CDS, are also found in the human sequence (e.g. BC032542) while the second one has evolved into a leucine. Although the beginning of the human CDS has been proposed to start from the position that corresponds to the third ATG in the murine sequence [22], we suggest that the first ATG would be a better starting codon than the third one; indeed, it is predicted to be the first triplet in the CDS by several gene prediction tools, e.g. GENSCAN analysis tool [28]. Further- more, a high degree of sequence similarity exists between the mouse and the human cDNA sequences upstream of the third ATG, suggesting that this segment is part of the CDS. In conclusion, we propose that the CDS starts not at the third but at the first ATG in the murine sequence, and that the human CDS starts at the corresponding position. Thus, we suggest that the human CDS of L -fucokinase becomes 94 amino acids longer than the corresponding CDS in the previous study [22]. L -fucose is a fundamental component of many mamma- lian glycoproteins and glycolipids. Fucosylation requires GDP- L -fucose as a donor of L -fucose, and a specific fucosyltransferase to catalyze the transfer of L -fucose to the acceptor molecules. The synthesis of GDP- L -fucose and its import into the Golgi lumen for a specific fucosyltrans- ferase is essential for selectin-dependent leukocyte traffick- ing and for normal human development. Leukocyte adhesion deficiency type 2 (LADII), also known as a congenital disorder of glycosylation IIc, is a rare human disorder of fucose metabolism in which the patient suffers from recurrent infection, persistent leukocytosis and severe mental and growth retardation [33,34]. Missense mutations in a Golgi-localized GDP-fucose transporter lead to parti- ally defective function and are responsible for the defective fucosylation in LADII patients [35,36]. Studies with LADII patients show that oral supplementation of fucose can restore selectin ligands and correct the immunodeficiency [37,38]. In this scenario, GDP- L -fucose is synthesized from oral fucose through the salvage pathway, which elevates the amountofGDP- L -fucose in the cytosol, leading to enhanced GDP-fucose uptake into the Golgi [35]. In a study by Smith et al. [39], the targeted disruption of the FX locus in the mouse ablates the de novo pathway for GDP- fucose synthesis from GDP-mannose causing adult animals to lack almost completely the fucosylated glycans in multiple tissues, leading to symptoms similar to those of LADII. The FX-deficient mice are completely dependent on dietary fucose, which restores the synthesis of GDP-fucose through the salvage pathway. The salvage metabolism accounts for approximately only 10% of the intracellular pool of GDP- L -fucose [40]. However, the enzymes of the salvage pathway are expressed with relatively high intensities in various animal tissues, e.g. brain, ovary, testis, kidney and liver, as shown by the quantitative real time PCR analysis in the present study and also in previous studies [21,23]. The wide expression of the enzymes involved in the salvage pathway of GDP- L -fucose can also be deduced from the large amount of data available from different EST libraries (e.g. http://www.ncbi.nlm.nih. gov/UniGene). Our analysis of the expression of the enzymes involved in the salvage pathway of GDP- L -fucose indicates that not only the de novo pathway alone, but also the salvage pathway could have an essential role in the synthesis of GDP- L -fucose in the cytosol. The importance and the regulatory mechanisms of the enzymes in the salvage pathway of GDP- L -fucose have not been elucidated, thus futher studies are needed. Acknowledgements We thank Tuula Kallioinen and Sirkka-Liisa Kauranen for skilled technical assistance in molecular biology, and Kati Vena ¨ la ¨ inen and Leena Penttila ¨ for assistance in HPLC and MALDI-TOF MS analysis. The work was supported in part by Research Grants of the Academy of Finland, the Technology Development Center (TEKES), Helsinki, the Sigrid Juselius Foundation, and the Helsinki University Central Hospital Fund (EVO). Fig. 8. Tissue expression patterns of murine L -fucokinase short and long splice variants and GDP- L -fucose pyrophosphorylase. The expression levels of the long splice variant of L -fucokinase (A), the short splice variant of L -fucokinase (B) and GDP- L -fucose pyrophosphorylase (C) were detected by quantitative real time PCR. The mRNA expression levels in each tissue were expressed relative to expression in the liver. Ó FEBS 2003 The salvage pathway of GDP- L -fucose in mouse (Eur. J. Biochem. 271)85 References 1. 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Cloning and expression of murine enzymes involved in the salvage pathway of GDP- L -fucose L -fucokinase and GDP- L -fucose pyrophosphorylase Jaana

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