1 Laboratory of Physiological Chemistry, ICP, and Université Catholique de Louvain, Brussels, Belgium
2 Brussels Branch of the Ludwig Institute, Brussels, Belgium
3 Center for Human Genetics, University of Leuven, Leuven, Belgium
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ABSTRACT |
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Primary amines of proteins and other compounds spontaneously react with carbohydrates that possess a free carbonyl group to produce Schiff bases. These slowly rearrange to form Amadori or Heyns products, depending on whether the reacting sugar was an aldose or a ketose (14). This process, known as nonenzymatic glycation, is best known for glucose, leading then to the production of fructosamines. Similar reactions occur with other sugars (mannose, galactose, fructose, and pentoses) (5) and phosphorylated sugar derivatives (68). In fact, glucose is intrinsically one of the least reactive sugars in this respect because it is well stabilized in its hemiacetalic form (5).
Formation of fructosamines has attracted much attention in the context of diabetes. This is because the glycation rate is first order with respect to glucose concentration. Serum fructosamines and glycated hemoglobin are therefore assayed to assess the blood glucose concentration in the preceding weeks or months (911). Furthermore, fructosamines are thought to participate in the pathogenesis of long-term diabetes complications by acting either as such (12) or after conversion to advanced glycation end products (1315).
The sequence of a mammalian protein that catalyzes the phosphorylation of low-molecular weight and protein-bound intracellular fructosamines on the third carbon of their deoxyfructose moiety has recently been reported (16,17). Because fructosamine 3-phosphate residues are unstable (17), fructosamine-3-kinase (FN3K) appears to cause the removal of fructosamine residues from proteins. Accordingly, 1-deoxy-1-morpholino-fructose, a (substrate and) competitive inhibitor of FN3K, caused a twofold increase in the accumulation of glycated hemoglobin in erythrocytes incubated with 200 mmol/l glucose (18).
While cloning the cDNA encoding FN3K, we noted the existence of human and mouse cDNAs encoding a related protein. In this study, we report the sequence of this FN3K-related protein (FN3K-RP) and the identification of its biochemical function.
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RESEARCH DESIGN AND METHODS |
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[14C]1-deoxy-1-morpholino-ribulose was synthesized by incubating 12.5 µCi [1-14C]ribose (American Radiochemical) with 30 µl morpholine at 75°C; 8 µl acetic acid and 40 µl ethanol were added after 30 and 40 min, respectively, and the incubation pursued for 15 min. Further purification on the cation exchanger (see above) resulted in a product that could be phosphorylated by FN3K or FN3K-RP to an extent of 50%. A more pure radiolabeled compound was obtained by purifying this product of phosphorylation with FN3K (obtained after a 40-min incubation at 30°C in the presence of 1.2 mU/ml FN3K, 5 mmol/l ATP-Mg, 25 mmol/l Tris, pH 7.8, 1 mmol/l EGTA, 1 mmol/l MgCl2, in a final volume of 10 ml) on an anion exchanger (AG1X8). The purified phosphorylation product was dephosphorylated with alkaline phosphatase, which was denatured with perchloric acid. More than 90% of the radioactive product could then be phosphorylated with FN3K or FN3K-RP.
For the synthesis of glycated lysozyme, a solution containing 60 mg/ml hen egg lysozyme, 25 mmol/l HEPES, pH 7.1, and 1 mol/l of the indicated aldoses was filtered on a 0.22-µm membrane and incubated at 37°C for 20 days (allose and glucose) or 3 days (ribose). Glycated lysozyme was purified by gel filtration on a Biogel P2 column equilibrated with water. The degree of glycation, estimated by nanoelectrospray mass spectrometry, was 1.35 mol/mol for glucose, 3.35 for allose, and 4 for ribose, which is consistent with the higher reactivity of allose, and most particularly of ribose, as compared with glucose (5).
Sequencing and preparation of expression vectors.
Human expressed sequence tag (EST) clones AI202129 and AI244490 were ordered from the U.K. Human Genome Mapping Project Resource Centre and sequenced (16). Rapid amplification of complementary DNA ends (RACE) (5' and 3') was carried out on mouse brain cDNA using the corresponding kits from Gibco BRL (Gaithersburg, MD). The amplification products were subcloned in pBluescript and sequenced. For the preparation of the bacterial expression vectors, a 5' primer containing the putative ATG codon in an NdeI site and a 3' primer containing the putative stop codon flanked by a BglII (human) or EcoRV (mouse) site were used to amplify human kidney or mouse brain cDNA with Pwo polymerase. The amplification products were subcloned in pBluescript, checked by sequencing, and inserted between the NdeI and BamHI sites of pET3a. For the preparation of the eukaryotic expression vector, the open-reading frame of human FN3K (16) and FN3K-RP, flanked with a perfect Kozak (22) consensus sequence (CCACCATGC), were PCR amplified with Pwo polymerase using the appropriate plasmids as templates and inserted between the EcoRI and BamHI sites (for FN3K) or EcoRI and XbaI sites (for FN3K-RP) of pCMV5.
Overexpression and purification of proteins.
HEK-293 cells were cultured under 5% CO2 at 37°C in Dulbeccos minimal essential medium containing 10% (vol/vol) FCS in 10-cm diameter Sarstedt dishes coated with poly-L-lysine. They were transfected using a modified calcium phosphate procedure (23) with 7.5 µg pCMV5 DNA and incubated overnight at 37°C under 3% CO2. The medium was replaced with fresh Dulbeccos minimal essential medium containing 10% (vol/vol) FCS, and the incubation was pursued at 37°C in the presence of 5% CO2. Cells were harvested after 48 h in 0.75 ml of a buffer containing 25 mmol/l HEPES, pH 7.1, 5 mg/l leupeptin, 5 mg/l antipain, and 0.5 mmol/l phenylmethylsulfonyl fluoride. After three cycles of freezing and thawing, the cell lysates were centrifuged for 30 min at 20,000g and 4°C.
The supernatant obtained from 20 dishes (
175 mg protein) was diluted with two volumes of buffer A (25 mmol/l HEPES, pH 7.1, 5 mg/l leupeptin, and 5 mg/l antipain) and loaded onto a 5-ml Blue Sepharose column at 4°C. The column was washed with 5 ml of buffer A and then with increasing NaCl concentration in buffer A. Protein was measured according to Bradford (24) with bovine
-globulin as a standard.
For the Western blots, proteins were separated in 10% SDS-polyacrylamide gels, transferred onto nitrocellulose membranes, and probed with rabbit polyclonal antibodies directed against recombinant human FN3K at a 1/1,000 dilution and then with horseradish peroxidase-conjugated anti-rabbit IgG (Sigma) at a 1/2,000 dilution. Immunocomplexes were visualized with the enhanced chemiluminescence reaction (Amersham Pharmacia Biotech). The amount of FN3K-RP and FN3K was estimated by comparing the intensity of bands corresponding to these proteins in Coomassie Blue-stained SDS-PAGE gels with those of protein standards.
RNA extraction and Northern blotting.
Total RNA was isolated from tissues from male 30-g NMRI mice (from Charles River) using the Ambion RNA-WIZ kit. Northern blots were performed (25) on 25 µg total RNA for all tissues, except for lenses, for which only 0.5 µg could be loaded. The probes used were the PCR-amplification products of the open-reading frames of mouse FN3K and FN3K-RP labeled with [
-32P] dCTP by random priming.
Measurement of enzymatic activity.
Phosphorylation of 1-deoxy-1-morpholino-fructose, 1-deoxy-1-morpholino-psicose, and psicoselysine was assayed at 30°C in a mixture containing 25 mmol/l Tris, pH 7.8, 1 mmol/l EGTA, 1 mmol/l MgCl2, 1 mmol/l dithiothreitol, 50 µmol/l ATP-Mg, and 500,000 cpm [-32P]ATP in a final volume of 60 µl. The reaction was stopped by adding 90 µl ice-cold 10% (w/vol) perchloric acid. After neutralization with K2CO3, the supernatants were diluted to 1 ml with 20 mmol/l MES, pH 6, and loaded onto anion-exchange columns (AG1X8, Cl- form, 1 ml). These were then washed with five volumes of 20 mmol/l MES, pH 6, to elute the phosphorylated Amadori compound, the unreacted [
-32P]ATP remaining bound to the column. The eluate was mixed with 15 ml OptimaGold (Packard) scintillation fluid and counted for radioactivity. Phosphorylation of glycated lysozyme was assayed by incorporation of 32P from [
-32P]ATP (16) in a mixture containing 25 mmol Tris, pH 7.8, 1 mmol/l EGTA, 1 mmol/l MgCl2, 1 mmol/l dithiothreitol, 2.1 mg/ml glycated hen egg lysozyme, and 100 µmol/l ATP-Mg. Phosphorylation of 1-deoxy-1-morpholino-ribulose was measured using [14C]1-deoxy-1-morpholino-ribulose, as previously described for [14C]1-deoxy-1-morpholino-fructose (16).
Preparation of phosphorylated 1-deoxy-1-morpholino-psicose for nuclear magnetic resonance and mass spectrometry analysis.
1-Deoxy-1-morpholino-psicose (25 µmol) was incubated for 18 h at 30°C in a final volume of 10 ml in the presence of 25 mmol/l Tris, pH 7.8, 1 mmol/l EGTA, 1 mmol/l dithiothreitol, 2 mmol/l ATP-Mg, 0.5 mg/ml BSA, and 1.1 mU of partially purified human recombinant FN3K-RP. The reaction medium was mixed with 2 ml ice-cold 70% (vol/vol) HClO4 and centrifuged for 20 min at 16,000g and 4°C. The supernatant was brought to neutral pH by mixing with 50 ml of a tris-N-octylamine/chloroform (1/3.6) mixture. The aqueous phase was diluted in water to a final volume of 45 ml and applied onto a 25-cm3 AG1X8 (Cl-) column, which was washed with 70 ml water to elute unreacted 1-deoxy-1-morpholino-psicose, and a linear NaCl gradient (2 x 125 ml; 00.5 mol/l NaCl) was applied to elute 1-deoxy-1-morpholino-psicose-phosphate. Fractions of 3 ml were collected, and the reducing power was measured (21). Fractions containing 1-deoxy-1-morpholino-psicose-phosphate were pooled and concentrated under vacuum to 2 ml and applied on a BiogelP2 column equilibrated in water to remove salts.
Mass spectrometry analysis was carried out as previously described (26). For the nuclear magnetic resonance (NMR) analysis, the preparation of 1-deoxy-1-morpholino-psicose-phosphate was evaporated to dryness, and the residue was dissolved in deuterated water. The pH of the final solution was 6.0. All NMR spectra were acquired on a Bruker DRX500 spectrometer at 30°C using a 5-mm probe head for inversion detection. Standard Bruker pulse programs were used to obtain one-dimensional phosphorus spectra (with and without proton decoupling), proton spectra (with and without phosphorus decoupling), 1H-1H correlation spectra (COSY), and 1H-31P-spectra (heteronuclear multiple bond connectivity [HMBC]). A delay of 50 ms was used for evolution of 3JPH constants. 31P chemical shifts were referenced to external 85% H3PO4 and proton chemical shifts and to 3-(trimethylsilyl)propanesulfonic acid (sodium salt).
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RESULTS |
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The mouse cDNA sequence encoding FN3K-RP was established by 5' and 3' RACE experiments starting from EST sequence AI647859, which contains nucleotides 445646 of the final cDNA sequence. The sequence that we obtained contained 48 bp of the 5' untranslated region and 78 bp of the 3' untranslated region (whose length is 2 kb, see below). It encoded a protein identical to hypothetical protein XP-137924, which in the meantime had appeared in databanks.
The predicted human and mouse FN3K-RPs have the same length (309 amino acids) as human and mouse FN3Ks (Fig. 1). They share 88% sequence identity with each other and 64% sequence identity with human and mouse FN3Ks. Like FN3Ks, their second amino acid is a glutamate, which suggests that their NH2-terminal residue is an acetylated methionine (27), as is the case for FN3K (16). They are homologous to bacterial proteins of unknown function, with which they share 2535% sequence identity (not shown). Like FN3K (16), FN3K-RP comprises an HGDxxxxN motif also found in aminoglycoside kinases.
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Two FN3K pseudogene fragments were identified on chromosome 22. One corresponds to exon 2 flanked by parts of introns 1 and 2 and the other to exon 3, intron 3, and exon 4, with adjacent parts of introns 2 and 4. They both share >95% identity with the human FN3K gene, indicating recent duplication events of portions of the FN3K gene.
As Szwergold, Howell, and Beisswenger (17) reported the localization of the FN3K gene on chromosome 1, we checked the chromosomal localization by fluorescence in-situ hybridization (FISH). For this, a PAC clone containing the FN3K gene (no. 127J18) was identified on human RPCI-1 PAC filters (28) by hybridization of a human FN3K probe. PCR amplification of introns using exonic primers showed that this clone contained both FN3K and FN3K-RP genes (not shown). FISH analysis indicated that it hybridized with the telomeric region of chromosome 17 (Fig. 2). No signal was detected on chromosome 1.
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We therefore resorted to expression in eukaryotic cells. Extracts of HEK cells that had been transfected with plasmids driving the expression of human FN3K or human FN3K-RP were chromatographed on Blue Sepharose, which is known to strongly retain FN3K (16). Proteins were eluted from the column with a salt gradient. Analysis of the fractions by Western blotting with anti-FN3K antibodies indicated the presence of FN3K in the fractions eluted with a high salt concentration from the column loaded with the extracts of cells expressing human FN3K (Fig. 4A). A protein eluting with lower salt concentrations was detected with the same antibodies in the fractions of the column loaded with FN3K-RP (Fig. 4B). None of these proteins could be detected in fractions of a column on which an extract of cells transfected with a control plasmid had been loaded (not shown). These results indicated that soluble FN3K-RP was expressed in HEK cells and that this protein had a distinct chromatographic behavior as compared with FN3K. Coomassie Blue staining of SDS-PAGE gels indicated that this protein accounted for 10% of total protein in fractions 4 to 6 of the column shown in Fig. 4A.
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These findings prompted us to study the phosphorylation of low-molecular weight psicosamines and ribulosamines. Both FN3K and FN3K-RP were found to phosphorylate 1-deoxy-1-morpholino-psicose, psicoselysine, and 1-deoxy-1-morpholino-ribulose, but only FN3K phosphorylated 1-deoxy-1-morpholino-fructose (Table 1) and fructoselysine (not shown). With both enzymes, the KM values for the model ribulosamine were 40- to 80-fold lower than for the low-molecular weight psicosamines, but only about 3- to 4-fold lower than the KM for 1-deoxy-1-morpholino-fructose in the case of FN3K. Furthermore, the latter enzyme displayed an 10-fold higher Vmax with 1-deoxy-1-morpholino-fructose than with 1-deoxy-1-morpholino-ribulose.
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Identification of the phosphorylated carbon.
To determine the identity of the carbon phosphorylated by FN3K-RP, 1-deoxy-1-morpholino-psicose was phosphorylated with this enzyme, purified, and analyzed by tandem mass spectrometry and NMR spectroscopy (Fig. 5). The first technique indicated that the purified product contained a major anion of m/z 328.1, which corresponds to the mass expected for 1-deoxy-1-morpholino-psicose-phosphate. Fragmentation of the latter yielded fragments of m/z 97.1 (corresponding to a phosphate group), 238.2 (corresponding to the loss of C4-C6 of the sugar moiety), and 241.1 (corresponding to the loss of the morpholine moiety). As in the case of 1-deoxy-1-morpholino-fructose 3-phosphate (16), the fact that C4-C6 can be removed without loss of the phosphate group indicates that phosphate is esterified to carbon 3.
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DISCUSSION |
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Another new finding reported in the present article is that FN3K has a much broader substrate specificity than previously thought because it acts not only on fructosamines but also on other ketosamines, with comparable (ribulosamines) or significantly lower affinities (psicosamines and maybe also tagatosamines, which were found to act as inhibitors) than it displays for fructosamines. This enzyme is therefore also a ketosamine- 3-kinase. It is, however, the only enzyme that acts on fructosamines, which are probably quantitatively the most important ketosamines that are formed under physiological conditions. Furthermore, it displays a higher catalytic efficiency (Vmax/KM ratio) on a model fructosamine (1-deoxy-1-morpholino-fructose) than on the equivalent ribulosamine (1-deoxy-1-morpholino-ribulose). Therefore, its name "fructosamine-3-kinase" appears justified, although its designation as "ketosamine-3-kinase 1" and that of FN3K-RP as "ketosamine-3-kinase 2" would be more rigorous. For the sake of clarity, the names FN3K and FN3K-RP will be used in the rest of the DISCUSSION.
Chromosomal localization and tissular distribution.
The gene encoding FN3K-RP is located next to the one encoding FN3K in both the human and mouse genomes, indicating an ancestral duplication event. The localization on human chromosome 17, as now reported in databases, was confirmed by in-situ hydridization with a PAC clone containing both genes. The previous localization of the FN3K gene on chromosome 1 (17) is most likely due to uncertain assignment of some PAC clones in an earlier phase of the human genome sequencing project.
Previous RT-PCR experiments performed on human RNAs indicated a widespread tissular distribution of FN3K, with particularly high levels in kidney (17). The Northern blots performed on mouse tissues in the present study indicate a wide variability of expression level from tissue to tissue. The most intense signals were observed with RNA from bone marrow, brain, and kidney, whereas other organs such as liver, intestinal mucosa, testis, thymus, and lung have no or barely detectable mRNA. It is not known at present if this difference in tissular distribution is due to a species difference or to a difference in the technique that was used.
Physiological function.
Recent work indicates that the function of FN3K is most likely to remove fructosamine residues from proteins (17,18). Fructosamine 3-phosphates are indeed unstable and decompose slowly to 3-deoxyglucosone, inorganic phosphate, and an amine (18). FN3K therefore appears to be a protein-repair enzyme. Such enzymes are expected to play an important role in tissues that contain proteins with long (half-)lives. This is certainly the case of erythrocytes and lenses (29), but it is also true for brain, where myelin proteins have a half-life much longer than 10 days (30). Interestingly, the knockout of another protein-repair enzyme, isoaspartyl-methyl-transferase (31), resulted in intractable seizures, leading to death of the mice after a few weeks. This result underlines the importance of protein-repair mechanisms in the brain and is consistent with the finding of elevated mRNA levels for both FN3K and FN3K-RP in this tissue.
As all ketosamine 3-phosphates other than fructosamine 3-phosphates are most likely also unstable, FN3K-RP could also potentially play the role of a deglycating enzyme. The most puzzling question is that of the identity of the substrates upon which this enzyme is acting physiologically. Its best in vitro substrates are two compounds with a hydroxyl group in the D configuration on C3 (D-ribulosamines and D-psicoamines), whereas it displays little if any affinity for ketosamines with an L configuration in C3. Fructosamines are indeed neither substrates nor inhibitors of FN3K-RP, and a tagatosamine and a xylulosamine were found to be poor inhibitors of this enzyme.
The question then is to know if and how these substrates could arise in vivo. As allose is not a physiological sugar, it cannot be a source of psicosamines. These could, however, arise through epimerization of fructosamines or of an intermediate in the formation of Amadori products from glucose (Fig. 7). Preliminary data indicate that when proteins are incubated with elevated concentrations of glucose in the presence of 200 mmol/l inorganic phosphate, which facilitates the formation of fructosamines (32), a substrate of FN3K-RP forms (F.C., E.V.S., unpublished data), suggesting that epimerization around the third carbon of fructosamines takes place under these conditions.
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Further evidence for a role of the new enzyme in combating glucose-induced glycation is to be found in the observation that the incubation of erythrocytes with an elevated glucose concentration under the conditions previously described (18) leads to the formation of substrates not only for FN3K but also FN3K-RP (G.D., F.C., J. Fortpied, and E.V.S., unpublished data). The identity of the FN3K-RP substrates formed under these conditions still remains to be determined; psicosamines and ribulosamines are both likely candidates.
Compared with FN3K-RP, FN3K has the unique capacity to phosphorylate fructosamines, suggesting that one of its important functions is to repair damages provoked by glucose. As both ketosamine-3-kinases phosphorylate psicosamines and ribulosamines with similar affinities, one may wonder if FN3K-RP has any specific role to play. One possibility is that this enzyme is able to phosphorylate as yet unknown substrates that are not utilizable by FN3K. In addition, it should be stressed that FN3K-RP appears to be expressed in tissues (testis and lung) where the FN3K mRNA is undetectable. In conclusion, the data reported in the present article indicate the existence of an enzyme that may complement the action of FN3K in a putative deglycation process.
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ACKNOWLEDGMENTS |
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The authors thank Geneviève Berghenouse for her help in experimental work, Helena Santos (Lisbon) for running and interpreting the NMR spectra, Didier Vertommen for his help in mass spectrometry analysis, and Maria Veiga-da-Cunha for her thoughtful comments.
GenBank accession nos.: AY360465, human fructosamine-3-kinase-related protein (human FN3K-RP); AY360466, mouse fructosamine-3-kinase-related protein (mouse FN3K-RP).
Address correspondence and reprint requests to E. Van Schaftingen, Avenue Hippocrate 75, B-1200, Brussels, Belgium. E-mail: vanschaftingen{at}bchm.ucl.ac.be
Received for publication March 10, 2003 and accepted in revised form September 15, 2003
COSY, correlation spectra; EST, expressed sequence tag; FISH, fluorescence in-situ hybridization; FN3K, fructosamine-3-kinase; FN3K-RP, FN3K-related protein; HEK, human embryonic kidney; HMBC, heteronuclear multiple bond connectivity; NMR, nuclear magnetic resonance; RACE, rapid amplification of complementary DNA ends
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REFERENCES |
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