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Emile Van Schaftingen - One of the best experts on this subject based on the ideXlab platform.
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many Fructosamine 3 kinase homologues in bacteria are ribulosamine erythrulosamine 3 kinases potentially involved in protein deglycation
FEBS Journal, 2007Co-Authors: Rita Gemayel, Didier Vertommen, Juliette Fortpied, Rim Rzem, Maria Veigadacunha, Emile Van SchaftingenAbstract:The purpose of this work was to identify the function of bacterial homologues of Fructosamine 3-kinase (FN3K), a mammalian enzyme responsible for the removal of Fructosamines from proteins. FN3K homologues were identified in approximately 200 (i.e. approximately 27%) of the sequenced bacterial genomes. In 11 of these genomes, from phylogenetically distant bacteria, the FN3K homologue was immediately preceded by a low-molecular-weight protein-tyrosine-phosphatase (LMW-PTP) homologue, which is therefore probably functionally related to the FN3K homologue. Five bacterial FN3K homologues (from Escherichia coli, Enterococcus faecium, Lactobacillus plantarum, Staphylococcus aureus and Thermus thermophilus) were overexpressed in E. coli, purified and their kinetic properties investigated. Four were ribulosamine/erythrulosamine 3-kinases acting best on free lysine and cadaverine derivatives, but not on ribulosamines bound to the alpha amino group of amino acids. They also phosphorylated protein-bound ribulosamines or erythrulosamines, but not protein-bound Fructosamines, therefore having properties similar to those of mammalian FN3K-related protein. The E. coli FN3K homologue (YniA) was inactive on all tested substrates. The LMW-PTP of T. thermophilus, which forms an operon with an FN3K homologue, and an LMW-PTP of S. aureus (PtpA) were overexpressed in E. coli, purified and shown to dephosphorylate not only protein tyrosine phosphates, but protein ribulosamine 5-phosphates as well as free ribuloselysine 5-phosphate and erythruloselysine 4-phosphate. These LMW-PTPs were devoid of ribulosamine 3-phosphatase activity. It is concluded that most bacterial FN3K homologues are ribulosamine/erythrulosamine 3-kinases. They may serve, in conjunction with a phosphatase, to deglycate products of glycation formed from ribose 5-phosphate or erythrose 4-phosphate.
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increased protein glycation in Fructosamine 3 kinase deficient mice
Biochemical Journal, 2006Co-Authors: Maria Veiga Dacunha, Patrick Jacquemin, Ghislain Delpierre, Catherine Godfraind, Ivan Théate, Didier Vertommen, Frédéric Clotman, Frédéric Lemaigre, Olivier Devuyst, Emile Van SchaftingenAbstract:Amines, including those present on proteins, spontaneously react with glucose to form Fructosamines in a reaction known as glycation. In the present paper, we have explored, through a targeted gene inactivation approach, the role of FN3K (Fructosamine 3-kinase), an intracellular enzyme that phosphorylates free and protein-bound fructose-epsilon-lysines and which is potentially involved in protein repair. Fn3k-/- mice looked healthy and had normal blood glucose and serum Fructosamine levels. However, their level of haemoglobin-bound Fructosamines was approx. 2.5-fold higher than that of control (Fn3k+/+) or Fn3k+/- mice. Other intracellular proteins were also significantly more glycated in Fn3k-/- mice in erythrocytes (1.8-2.2-fold) and in brain, kidney, liver and skeletal muscle (1.2-1.8-fold), indicating that FN3K removes Fructosamines from intracellular proteins in vivo. The urinary excretion of free fructose-epsilon-lysine was 10-20-fold higher in fed mice compared with mice starved for 36 h, and did not differ between fed Fn3k+/+ and Fn3k-/- mice, indicating that food is the main source of urinary fructose-epsilon-lysine in these mice and that FN3K does not participate in the metabolism of food-derived fructose-epsilon-lysine. However, in starved animals, the urinary excretion of fructose-epsilon-lysine was 2.5-fold higher in Fn3k-/- mice compared with Fn3k+/+ or Fn3k+/- mice. Furthermore, a marked increase (5-13-fold) was observed in the concentration of free fructose-epsilon-lysine in tissues of fed Fn3k-/- mice compared with control mice, indicating that FN3K participates in the metabolism of endogenously produced fructose-epsilon-lysine. Taken together, these data indicate that FN3K serves as a protein repair enzyme and also in the metabolism of endogenously produced free fructose-epsilon-lysine.
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magnesium dependent phosphatase 1 is a protein Fructosamine 6 phosphatase potentially involved in glycation repair
Journal of Biological Chemistry, 2006Co-Authors: Juliette Fortpied, Didier Vertommen, Pushpa Maliekal, Emile Van SchaftingenAbstract:Fructosamine-3-kinase (FN3K) is a recently described protein-repair enzyme responsible for the removal of Fructosamines, which are the products of a spontaneous reaction of glucose with amines. We show here that, compared with glucose, glucose 6-phosphate (Glu-6-P) reacted 3-6-fold more rapidly with proteins and 8-fold more rapidly with N-alpha-t-Boc-lysine, being therefore a more significant intracellular glycating agent than glucose in skeletal muscle and heart. Fructosamine 6-phosphates, which result from the reaction of amines with Glu-6-P, were not substrates for FN3K. However, a phosphatase that dephosphorylates protein-bound Fructosamine 6-phosphates was found to be present in rat tissues. This enzyme was purified to near homogeneity from skeletal muscle and was identified as magnesium-dependent phosphatase-1 (MDP-1), an enzyme of the haloacid dehalogenase family with a putative protein-tyrosine phosphatase function. Human recombinant MDP-1 acted on protein-bound Fructosamine 6-phosphates with a catalytic efficiency >10-fold higher than those observed with its next best substrates (arabinose 5-phosphate and free fructoselysine 6-phosphate) and >100-fold higher than with protein-phosphotyrosine. It had no detectable activity on Fructosamine 3-phosphates. MDP-1 dephosphorylated up to approximately 75% of the Fructosamine 6-phosphates that are present on lysozyme after incubation of this protein with Glu-6-P. Furthermore, lysozyme glycated with Glu-6-P was converted by MDP-1 to a substrate for FN3K. We conclude that MDP-1 may act physiologically in conjunction with FN3K to free proteins from the glycation products derived from Glu-6-P.
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Increased protein glycation in Fructosamine-3-kinase-deficient mice
Biochemical Journal, 2006Co-Authors: Maria Veiga-da-cunha, Patrick Jacquemin, Ghislain Delpierre, Catherine Godfraind, Ivan Théate, Didier Vertommen, Frédéric Clotman, Frédéric Lemaigre, Olivier Devuyst, Emile Van SchaftingenAbstract:Amines, including those present on proteins, spontaneously react with glucose to form Fructosamines in a reaction known as glycation. Here, we explored through a targeted gene inactivation approach, the role of Fructosamine-3-kinase (FN3K), an intracellular enzyme that phosphorylates free and protein-bound fructose-{epsilon}-lysines and which is potentially involved in protein repair. FN3K -/-} mice looked healthy and had normal blood glucose and serum Fructosamine levels. However, their level of hemoglobin-bound Fructosamines was about 2.5-fold higher than that of control or FN3K +/-} mice. Other intracellular proteins were also significantly more glycated in FN3K -/-} mice in erythrocytes (1.8 to 2.2-fold) and in brain, kidney, liver and skeletal muscle (1.2 to 1.8-fold), indicating that, in vivo, FN3K removes Fructosamines from intracellular proteins. The urinary excretion of free fructose-{epsilon}-lysine was 10-20 fold higher in fed mice compared to 36 h-starved mice and did not differ between fed FN3K +/+} and FN3K -/-} mice, indicating that food is the main source of urinary fructose-{epsilon}-lysine in these mice and that FN3K does not participate in the metabolism of food-derived fructose-{epsilon}-lysine. However, in starved animals, the urinary excretion of fructose-{epsilon}-lysine was 2.5-fold higher in FN3K -/-} mice than in FN3K +/+} or FN3K +/-} mice. Furthermore, a marked increase (5 to 13-fold) was observed in the concentration of free fructose-{epsilon}-lysine in tissues of fed FN3K -/-} mice compared to control mice, indicating that FN3K participates in the metabolism of endogenously-produced fructose-{epsilon}-lysine. Taken together, these data indicate that FN3K serves both as a protein repair enzyme and in the metabolism of endogenously produced free fructose-{epsilon}-lysine.
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tissue distribution and evolution of Fructosamine 3 kinase and Fructosamine 3 kinase related protein
Journal of Biological Chemistry, 2004Co-Authors: Jerome Delplanque, Ghislain Delpierre, Frederik Opperdoes, Emile Van SchaftingenAbstract:Fructosamine 3-kinase (FN3K) and FN3K-related protein (FN3K-RP) catalyze the phosphorylation of the Amadori products ribulosamines, psicosamines, and, in the case of FN3K, Fructosamines. BLAST searches in chordate genomes revealed two genes encoding proteins homologous to FN3K or FN3K-RP in various mammals and in chicken but only one gene, encoding a protein more similar to FN3K-RP than to FN3K, in fishes and the sea squirt Ciona intestinalis. This suggests that a gene duplication event occurred after the fish radiation and that the FN3K gene evolved more rapidly than the FN3K-RP gene. In agreement with this distribution, only one enzyme, phosphorylating ribulosamines and psicosamines but not Fructosamines, was found in the tissues from a fish (Clarias gariepinus), whereas two enzymes with specificities similar to either FN3K or FN3K-RP were found in mouse, rat, and chicken tissues. FN3K is particularly active in brain, heart, kidney, and skeletal muscle. Its activity is also relatively elevated in erythrocytes from man, rat, and mouse but barely detectable in erythrocytes from chicken and pig, which correlates well with the low intracellular concentration of glucose in erythrocytes from these species. This is in keeping with the specific role of FN3K to repair protein damage caused by glucose. FN3K-RP was more evenly distributed in tissues, except for skeletal muscle where its activity was particularly low. This may be related to low activity of the pentose phosphate pathway in this tissue, as suggested by assays of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. This finding, together with the high affinity of FN3K-RP for ribulosamines, suggests that this enzyme may serve to repair damage caused by the powerful glycating agent, ribose 5-phosphate.
François Collard - One of the best experts on this subject based on the ideXlab platform.
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Enzymatic repair of Amadori products
Amino Acids, 2012Co-Authors: Emile Schaftingen, François Collard, Elsa Wiame, Maria Veiga-da-cunhaAbstract:Protein deglycation, a new form of protein repair, involves several enzymes. Fructosamine-3-kinase (FN3K), an enzyme found in mammals and birds, phosphorylates Fructosamines on the third carbon of their sugar moiety, making them unstable and causing them to detach from proteins. This enzyme acts particularly well on fructose-epsilon-lysine, both in free form and in the accessible regions of proteins. Mice deficient in FN3K accumulate protein-bound Fructosamines and free fructoselysine, indicating that the deglycation mechanism initiated by FN3K is operative in vivo. Mammals and birds also have an enzyme designated ‘FN3K-related protein’ (FN3KRP), which shares ≈65% sequence identity with FN3K. Unlike FN3K, FN3KRP does not phosphorylate Fructosamines, but acts on ribulosamines and erythrulosamines. As with FN3K, the third carbon is phosphorylated and this leads to destabilization of the ketoamines. Experiments with intact erythrocytes indicate that FN3KRP is also a protein-repair enzyme. Its physiological substrates are most likely formed from ribose 5-phosphate and erythrose 4-phosphate, which give rise to ketoamine 5- or 4-phosphates. The latter are dephosphorylated by ‘low-molecular-weight protein-tyrosine-phosphatase-A’ (LMW-PTP-A) before FN3KRP transfers a phosphate on the third carbon. The specificity of FN3K homologues present in plants and bacteria is similar to that of mammalian FN3KRP, suggesting that deglycation of ribulosamines and/or erythrulosamines is an ancient mechanism. Mammalian cells contain also a phosphatase acting on Fructosamine 6-phosphates, which result from the reaction of proteins with glucose 6-phosphate.
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the cation π interaction between lys53 and the flavin of Fructosamine oxidase faox ii is critical for activity
Biochemistry, 2011Co-Authors: François Collard, Jianye Zhang, Ina Nemet, Rebecca L Fagan, Bruce A Palfey, Vincent M MonnierAbstract:Fructosamine oxidases (FAOXs) are flavin-containing enzymes that catalyze the oxidative deglycation of low molecular weight Fructosamines or Amadori products. The Fructosamine substrate is oxidized by the flavin in the reductive half-reaction, and the reduced flavin is then oxidized by molecular oxygen in the oxidative half-reaction. The crystal structure of FAOX-II from Aspergillus fumigatus reveals a unique interaction between Lys53 and the isoalloxazine. The ammonium nitrogen of the lysine is in contact with and nearly centered over the aromatic ring of the flavin on the si-face. Here, we investigate the importance of this unique interaction on the reactions catalyzed by FAOX by studying both half-reactions of the wild-type and Lys53 mutant enzymes. The positive charge of Lys53 is critical for flavin reduction but plays very little role in the reaction with molecular oxygen. The conservative mutation of Lys53 to arginine had minor effects on catalysis. However, removing the charge by replacing Lys53 wi...
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crystal structure of the deglycating enzyme Fructosamine oxidase amadoriase ii
Journal of Biological Chemistry, 2008Co-Authors: François Collard, Jianye Zhang, Ina Nemet, Kaustubha R Qanungo, Vincent M Monnier, Vivien C YeeAbstract:Fructosamine oxidases (FAOX) catalyze the oxidative deglycation of low molecular weight Fructosamines (Amadori products). These proteins are of interest in developing an enzyme to deglycate proteins implicated in diabetic complications. We report here the crystal structures of FAOX-II from the fungi Aspergillus fumigatus, in free form and in complex with the inhibitor fructosyl-thioacetate, at 1.75 and 1.6A resolution, respectively. FAOX-II is a two domain FAD-enzyme with an overall topology that is most similar to that of monomeric sarcosine oxidase. Active site residues Tyr-60, Arg-112 and Lys-368 bind the carboxylic portion of the Fructosamine, whereas Glu-280 and Arg-411 bind the fructosyl portion. From structure-guided sequence comparison, Glu-280 was identified as a signature residue for FAOX activity. Two flexible surface loops become ordered upon binding of the inhibitor in a catalytic site that is about 12A deep, providing an explanation for the very low activity of FAOX enzymes toward protein-bound Fructosamines, which would have difficulty accessing the active site. Structure-based mutagenesis showed that substitution of Glu-280 and Arg-411 eliminates enzyme activity. In contrast, modification of other active site residues or of amino acids in the flexible active site loops has little effect, highlighting these regions as potential targets in designing an enzyme that will accept larger substrates.
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Fructosamine 3 kinase related protein and deglycation in human erythrocytes
Biochemical Journal, 2004Co-Authors: François Collard, Elsa Wiame, Ghislain Delpierre, Didier Vertommen, Juliette Fortpied, Niki Bergans, Florent Vanstapel, Emile Van SchaftingenAbstract:Fructosamine 3-kinase (FN3K), an enzyme initially identified in erythrocytes, catalyses the phosphorylation of Fructosamines on their third carbon, leading to their destabilization and their removal from protein. We show that human erythrocytes also contain FN3K-related protein (FN3K-RP), an enzyme that phosphorylates psicosamines and ribulosamines, but not Fructosamines, on the third carbon of their sugar moiety. Protein-bound psicosamine 3-phosphates and ribulosamine 3-phosphates are unstable, decomposing at pH 7.1 and 37 degrees C with half-lives of 8.8 h and 25 min respectively, as compared with 7 h for Fructosamine 3-phosphates. NMR analysis indicated that 1-deoxy-1-morpholinopsicose (DMP, a substrate for FN3K and FN3K-RP), like 1-deoxy-1-morpholinofructose (DMF, a substrate of FN3K), penetrated erythrocytes and was converted into the corresponding 3-phospho-derivative. Incubation of erythrocytes with 50 mM allose, 200 mM glucose or 10 mM ribose for 24 h resulted in the accumulation of glycated haemoglobin, and this accumulation was approx. 1.9-2.6-fold higher if DMP, a competitive inhibitor of both FN3K and FN3K-RP, was present in the incubation medium. Incubation with 50 mM allose or 200 mM glucose also caused the accumulation of ketoamine 3-phosphates, which was inhibited by DMP. By contrast, DMF, a specific inhibitor of FN3K, only affected the glucose-dependent accumulation of glycated haemoglobin and ketoamine 3-phosphates. These data indicate that FN3K-RP can phosphorylate intracellular, protein-bound psicosamines and ribulosamines, thus leading to deglycation.
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a mammalian protein homologous to Fructosamine 3 kinase is a ketosamine 3 kinase acting on psicosamines and ribulosamines but not on Fructosamines
Diabetes, 2003Co-Authors: François Collard, Ghislain Delpierre, Vincent Stroobant, Gert Matthijs, Emile Van SchaftingenAbstract:Fructosamine-3-kinase (FN3K) is an enzyme that appears to be responsible for the removal of Fructosamines from proteins. In this study, we report the sequence of human and mouse cDNAs encoding proteins sharing 65% sequence identity with FN3K. The genes encoding FN3K and FN3K-related protein (FN3K-RP) are present next to each other on human chromosome 17q25, and they both have a similar 6-exon structure. Northern blots of mouse tissues RNAs indicate a high level of expression of both genes in bone marrow, brain, kidneys, and spleen. Human FN3K-RP was transfected in human embryonic kidney (HEK) cells, and the expressed protein was partially purified by chromatography on Blue Sepharose. Unlike FN3K, FN3K-RP did not phosphorylate fructoselysine, 1-deoxy-1-morpholino-fructose, or lysozyme glycated with glucose. In a more systematic screening for potential substrates for FN3K-RP, we found, however, that both enzymes phosphorylated ketosamines with a D-configuration in C3 (psicoselysine, 1-deoxy-1-morpholino-psicose, 1-deoxy-1-morpholino-ribulose, lysozyme glycated with allose-the C3 epimer of glucose, or with ribose). Tandem mass spectrometry and nuclear magnetic resonance analysis of the product of phosphorylation of 1-deoxy-1-morpholino-psicose by FN3K-RP indicated that this enzyme phosphorylates the third carbon of the sugar moiety. These results indicate that FN3K-RP is a ketosamine-3-kinase (ketosamine-3-kinase 2). This enzyme presumably plays a role in freeing proteins from ribulosamines or psicosamines, which might arise in a several step process, from the reaction of amines with glucose and/or glycolytic intermediates. This role is shared by Fructosamine-3-kinase (ketosamine-3-kinase 1), which has, in addition, the unique capacity to phosphorylate Fructosamines.
Didier Vertommen - One of the best experts on this subject based on the ideXlab platform.
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many Fructosamine 3 kinase homologues in bacteria are ribulosamine erythrulosamine 3 kinases potentially involved in protein deglycation
FEBS Journal, 2007Co-Authors: Rita Gemayel, Didier Vertommen, Juliette Fortpied, Rim Rzem, Maria Veigadacunha, Emile Van SchaftingenAbstract:The purpose of this work was to identify the function of bacterial homologues of Fructosamine 3-kinase (FN3K), a mammalian enzyme responsible for the removal of Fructosamines from proteins. FN3K homologues were identified in approximately 200 (i.e. approximately 27%) of the sequenced bacterial genomes. In 11 of these genomes, from phylogenetically distant bacteria, the FN3K homologue was immediately preceded by a low-molecular-weight protein-tyrosine-phosphatase (LMW-PTP) homologue, which is therefore probably functionally related to the FN3K homologue. Five bacterial FN3K homologues (from Escherichia coli, Enterococcus faecium, Lactobacillus plantarum, Staphylococcus aureus and Thermus thermophilus) were overexpressed in E. coli, purified and their kinetic properties investigated. Four were ribulosamine/erythrulosamine 3-kinases acting best on free lysine and cadaverine derivatives, but not on ribulosamines bound to the alpha amino group of amino acids. They also phosphorylated protein-bound ribulosamines or erythrulosamines, but not protein-bound Fructosamines, therefore having properties similar to those of mammalian FN3K-related protein. The E. coli FN3K homologue (YniA) was inactive on all tested substrates. The LMW-PTP of T. thermophilus, which forms an operon with an FN3K homologue, and an LMW-PTP of S. aureus (PtpA) were overexpressed in E. coli, purified and shown to dephosphorylate not only protein tyrosine phosphates, but protein ribulosamine 5-phosphates as well as free ribuloselysine 5-phosphate and erythruloselysine 4-phosphate. These LMW-PTPs were devoid of ribulosamine 3-phosphatase activity. It is concluded that most bacterial FN3K homologues are ribulosamine/erythrulosamine 3-kinases. They may serve, in conjunction with a phosphatase, to deglycate products of glycation formed from ribose 5-phosphate or erythrose 4-phosphate.
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increased protein glycation in Fructosamine 3 kinase deficient mice
Biochemical Journal, 2006Co-Authors: Maria Veiga Dacunha, Patrick Jacquemin, Ghislain Delpierre, Catherine Godfraind, Ivan Théate, Didier Vertommen, Frédéric Clotman, Frédéric Lemaigre, Olivier Devuyst, Emile Van SchaftingenAbstract:Amines, including those present on proteins, spontaneously react with glucose to form Fructosamines in a reaction known as glycation. In the present paper, we have explored, through a targeted gene inactivation approach, the role of FN3K (Fructosamine 3-kinase), an intracellular enzyme that phosphorylates free and protein-bound fructose-epsilon-lysines and which is potentially involved in protein repair. Fn3k-/- mice looked healthy and had normal blood glucose and serum Fructosamine levels. However, their level of haemoglobin-bound Fructosamines was approx. 2.5-fold higher than that of control (Fn3k+/+) or Fn3k+/- mice. Other intracellular proteins were also significantly more glycated in Fn3k-/- mice in erythrocytes (1.8-2.2-fold) and in brain, kidney, liver and skeletal muscle (1.2-1.8-fold), indicating that FN3K removes Fructosamines from intracellular proteins in vivo. The urinary excretion of free fructose-epsilon-lysine was 10-20-fold higher in fed mice compared with mice starved for 36 h, and did not differ between fed Fn3k+/+ and Fn3k-/- mice, indicating that food is the main source of urinary fructose-epsilon-lysine in these mice and that FN3K does not participate in the metabolism of food-derived fructose-epsilon-lysine. However, in starved animals, the urinary excretion of fructose-epsilon-lysine was 2.5-fold higher in Fn3k-/- mice compared with Fn3k+/+ or Fn3k+/- mice. Furthermore, a marked increase (5-13-fold) was observed in the concentration of free fructose-epsilon-lysine in tissues of fed Fn3k-/- mice compared with control mice, indicating that FN3K participates in the metabolism of endogenously produced fructose-epsilon-lysine. Taken together, these data indicate that FN3K serves as a protein repair enzyme and also in the metabolism of endogenously produced free fructose-epsilon-lysine.
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magnesium dependent phosphatase 1 is a protein Fructosamine 6 phosphatase potentially involved in glycation repair
Journal of Biological Chemistry, 2006Co-Authors: Juliette Fortpied, Didier Vertommen, Pushpa Maliekal, Emile Van SchaftingenAbstract:Fructosamine-3-kinase (FN3K) is a recently described protein-repair enzyme responsible for the removal of Fructosamines, which are the products of a spontaneous reaction of glucose with amines. We show here that, compared with glucose, glucose 6-phosphate (Glu-6-P) reacted 3-6-fold more rapidly with proteins and 8-fold more rapidly with N-alpha-t-Boc-lysine, being therefore a more significant intracellular glycating agent than glucose in skeletal muscle and heart. Fructosamine 6-phosphates, which result from the reaction of amines with Glu-6-P, were not substrates for FN3K. However, a phosphatase that dephosphorylates protein-bound Fructosamine 6-phosphates was found to be present in rat tissues. This enzyme was purified to near homogeneity from skeletal muscle and was identified as magnesium-dependent phosphatase-1 (MDP-1), an enzyme of the haloacid dehalogenase family with a putative protein-tyrosine phosphatase function. Human recombinant MDP-1 acted on protein-bound Fructosamine 6-phosphates with a catalytic efficiency >10-fold higher than those observed with its next best substrates (arabinose 5-phosphate and free fructoselysine 6-phosphate) and >100-fold higher than with protein-phosphotyrosine. It had no detectable activity on Fructosamine 3-phosphates. MDP-1 dephosphorylated up to approximately 75% of the Fructosamine 6-phosphates that are present on lysozyme after incubation of this protein with Glu-6-P. Furthermore, lysozyme glycated with Glu-6-P was converted by MDP-1 to a substrate for FN3K. We conclude that MDP-1 may act physiologically in conjunction with FN3K to free proteins from the glycation products derived from Glu-6-P.
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Increased protein glycation in Fructosamine-3-kinase-deficient mice
Biochemical Journal, 2006Co-Authors: Maria Veiga-da-cunha, Patrick Jacquemin, Ghislain Delpierre, Catherine Godfraind, Ivan Théate, Didier Vertommen, Frédéric Clotman, Frédéric Lemaigre, Olivier Devuyst, Emile Van SchaftingenAbstract:Amines, including those present on proteins, spontaneously react with glucose to form Fructosamines in a reaction known as glycation. Here, we explored through a targeted gene inactivation approach, the role of Fructosamine-3-kinase (FN3K), an intracellular enzyme that phosphorylates free and protein-bound fructose-{epsilon}-lysines and which is potentially involved in protein repair. FN3K -/-} mice looked healthy and had normal blood glucose and serum Fructosamine levels. However, their level of hemoglobin-bound Fructosamines was about 2.5-fold higher than that of control or FN3K +/-} mice. Other intracellular proteins were also significantly more glycated in FN3K -/-} mice in erythrocytes (1.8 to 2.2-fold) and in brain, kidney, liver and skeletal muscle (1.2 to 1.8-fold), indicating that, in vivo, FN3K removes Fructosamines from intracellular proteins. The urinary excretion of free fructose-{epsilon}-lysine was 10-20 fold higher in fed mice compared to 36 h-starved mice and did not differ between fed FN3K +/+} and FN3K -/-} mice, indicating that food is the main source of urinary fructose-{epsilon}-lysine in these mice and that FN3K does not participate in the metabolism of food-derived fructose-{epsilon}-lysine. However, in starved animals, the urinary excretion of fructose-{epsilon}-lysine was 2.5-fold higher in FN3K -/-} mice than in FN3K +/+} or FN3K +/-} mice. Furthermore, a marked increase (5 to 13-fold) was observed in the concentration of free fructose-{epsilon}-lysine in tissues of fed FN3K -/-} mice compared to control mice, indicating that FN3K participates in the metabolism of endogenously-produced fructose-{epsilon}-lysine. Taken together, these data indicate that FN3K serves both as a protein repair enzyme and in the metabolism of endogenously produced free fructose-{epsilon}-lysine.
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Fructosamine 3 kinase related protein and deglycation in human erythrocytes
Biochemical Journal, 2004Co-Authors: François Collard, Elsa Wiame, Ghislain Delpierre, Didier Vertommen, Juliette Fortpied, Niki Bergans, Florent Vanstapel, Emile Van SchaftingenAbstract:Fructosamine 3-kinase (FN3K), an enzyme initially identified in erythrocytes, catalyses the phosphorylation of Fructosamines on their third carbon, leading to their destabilization and their removal from protein. We show that human erythrocytes also contain FN3K-related protein (FN3K-RP), an enzyme that phosphorylates psicosamines and ribulosamines, but not Fructosamines, on the third carbon of their sugar moiety. Protein-bound psicosamine 3-phosphates and ribulosamine 3-phosphates are unstable, decomposing at pH 7.1 and 37 degrees C with half-lives of 8.8 h and 25 min respectively, as compared with 7 h for Fructosamine 3-phosphates. NMR analysis indicated that 1-deoxy-1-morpholinopsicose (DMP, a substrate for FN3K and FN3K-RP), like 1-deoxy-1-morpholinofructose (DMF, a substrate of FN3K), penetrated erythrocytes and was converted into the corresponding 3-phospho-derivative. Incubation of erythrocytes with 50 mM allose, 200 mM glucose or 10 mM ribose for 24 h resulted in the accumulation of glycated haemoglobin, and this accumulation was approx. 1.9-2.6-fold higher if DMP, a competitive inhibitor of both FN3K and FN3K-RP, was present in the incubation medium. Incubation with 50 mM allose or 200 mM glucose also caused the accumulation of ketoamine 3-phosphates, which was inhibited by DMP. By contrast, DMF, a specific inhibitor of FN3K, only affected the glucose-dependent accumulation of glycated haemoglobin and ketoamine 3-phosphates. These data indicate that FN3K-RP can phosphorylate intracellular, protein-bound psicosamines and ribulosamines, thus leading to deglycation.
Ghislain Delpierre - One of the best experts on this subject based on the ideXlab platform.
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increased protein glycation in Fructosamine 3 kinase deficient mice
Biochemical Journal, 2006Co-Authors: Maria Veiga Dacunha, Patrick Jacquemin, Ghislain Delpierre, Catherine Godfraind, Ivan Théate, Didier Vertommen, Frédéric Clotman, Frédéric Lemaigre, Olivier Devuyst, Emile Van SchaftingenAbstract:Amines, including those present on proteins, spontaneously react with glucose to form Fructosamines in a reaction known as glycation. In the present paper, we have explored, through a targeted gene inactivation approach, the role of FN3K (Fructosamine 3-kinase), an intracellular enzyme that phosphorylates free and protein-bound fructose-epsilon-lysines and which is potentially involved in protein repair. Fn3k-/- mice looked healthy and had normal blood glucose and serum Fructosamine levels. However, their level of haemoglobin-bound Fructosamines was approx. 2.5-fold higher than that of control (Fn3k+/+) or Fn3k+/- mice. Other intracellular proteins were also significantly more glycated in Fn3k-/- mice in erythrocytes (1.8-2.2-fold) and in brain, kidney, liver and skeletal muscle (1.2-1.8-fold), indicating that FN3K removes Fructosamines from intracellular proteins in vivo. The urinary excretion of free fructose-epsilon-lysine was 10-20-fold higher in fed mice compared with mice starved for 36 h, and did not differ between fed Fn3k+/+ and Fn3k-/- mice, indicating that food is the main source of urinary fructose-epsilon-lysine in these mice and that FN3K does not participate in the metabolism of food-derived fructose-epsilon-lysine. However, in starved animals, the urinary excretion of fructose-epsilon-lysine was 2.5-fold higher in Fn3k-/- mice compared with Fn3k+/+ or Fn3k+/- mice. Furthermore, a marked increase (5-13-fold) was observed in the concentration of free fructose-epsilon-lysine in tissues of fed Fn3k-/- mice compared with control mice, indicating that FN3K participates in the metabolism of endogenously produced fructose-epsilon-lysine. Taken together, these data indicate that FN3K serves as a protein repair enzyme and also in the metabolism of endogenously produced free fructose-epsilon-lysine.
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Increased protein glycation in Fructosamine-3-kinase-deficient mice
Biochemical Journal, 2006Co-Authors: Maria Veiga-da-cunha, Patrick Jacquemin, Ghislain Delpierre, Catherine Godfraind, Ivan Théate, Didier Vertommen, Frédéric Clotman, Frédéric Lemaigre, Olivier Devuyst, Emile Van SchaftingenAbstract:Amines, including those present on proteins, spontaneously react with glucose to form Fructosamines in a reaction known as glycation. Here, we explored through a targeted gene inactivation approach, the role of Fructosamine-3-kinase (FN3K), an intracellular enzyme that phosphorylates free and protein-bound fructose-{epsilon}-lysines and which is potentially involved in protein repair. FN3K -/-} mice looked healthy and had normal blood glucose and serum Fructosamine levels. However, their level of hemoglobin-bound Fructosamines was about 2.5-fold higher than that of control or FN3K +/-} mice. Other intracellular proteins were also significantly more glycated in FN3K -/-} mice in erythrocytes (1.8 to 2.2-fold) and in brain, kidney, liver and skeletal muscle (1.2 to 1.8-fold), indicating that, in vivo, FN3K removes Fructosamines from intracellular proteins. The urinary excretion of free fructose-{epsilon}-lysine was 10-20 fold higher in fed mice compared to 36 h-starved mice and did not differ between fed FN3K +/+} and FN3K -/-} mice, indicating that food is the main source of urinary fructose-{epsilon}-lysine in these mice and that FN3K does not participate in the metabolism of food-derived fructose-{epsilon}-lysine. However, in starved animals, the urinary excretion of fructose-{epsilon}-lysine was 2.5-fold higher in FN3K -/-} mice than in FN3K +/+} or FN3K +/-} mice. Furthermore, a marked increase (5 to 13-fold) was observed in the concentration of free fructose-{epsilon}-lysine in tissues of fed FN3K -/-} mice compared to control mice, indicating that FN3K participates in the metabolism of endogenously-produced fructose-{epsilon}-lysine. Taken together, these data indicate that FN3K serves both as a protein repair enzyme and in the metabolism of endogenously produced free fructose-{epsilon}-lysine.
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tissue distribution and evolution of Fructosamine 3 kinase and Fructosamine 3 kinase related protein
Journal of Biological Chemistry, 2004Co-Authors: Jerome Delplanque, Ghislain Delpierre, Frederik Opperdoes, Emile Van SchaftingenAbstract:Fructosamine 3-kinase (FN3K) and FN3K-related protein (FN3K-RP) catalyze the phosphorylation of the Amadori products ribulosamines, psicosamines, and, in the case of FN3K, Fructosamines. BLAST searches in chordate genomes revealed two genes encoding proteins homologous to FN3K or FN3K-RP in various mammals and in chicken but only one gene, encoding a protein more similar to FN3K-RP than to FN3K, in fishes and the sea squirt Ciona intestinalis. This suggests that a gene duplication event occurred after the fish radiation and that the FN3K gene evolved more rapidly than the FN3K-RP gene. In agreement with this distribution, only one enzyme, phosphorylating ribulosamines and psicosamines but not Fructosamines, was found in the tissues from a fish (Clarias gariepinus), whereas two enzymes with specificities similar to either FN3K or FN3K-RP were found in mouse, rat, and chicken tissues. FN3K is particularly active in brain, heart, kidney, and skeletal muscle. Its activity is also relatively elevated in erythrocytes from man, rat, and mouse but barely detectable in erythrocytes from chicken and pig, which correlates well with the low intracellular concentration of glucose in erythrocytes from these species. This is in keeping with the specific role of FN3K to repair protein damage caused by glucose. FN3K-RP was more evenly distributed in tissues, except for skeletal muscle where its activity was particularly low. This may be related to low activity of the pentose phosphate pathway in this tissue, as suggested by assays of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. This finding, together with the high affinity of FN3K-RP for ribulosamines, suggests that this enzyme may serve to repair damage caused by the powerful glycating agent, ribose 5-phosphate.
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Fructosamine 3 kinase related protein and deglycation in human erythrocytes
Biochemical Journal, 2004Co-Authors: François Collard, Elsa Wiame, Ghislain Delpierre, Didier Vertommen, Juliette Fortpied, Niki Bergans, Florent Vanstapel, Emile Van SchaftingenAbstract:Fructosamine 3-kinase (FN3K), an enzyme initially identified in erythrocytes, catalyses the phosphorylation of Fructosamines on their third carbon, leading to their destabilization and their removal from protein. We show that human erythrocytes also contain FN3K-related protein (FN3K-RP), an enzyme that phosphorylates psicosamines and ribulosamines, but not Fructosamines, on the third carbon of their sugar moiety. Protein-bound psicosamine 3-phosphates and ribulosamine 3-phosphates are unstable, decomposing at pH 7.1 and 37 degrees C with half-lives of 8.8 h and 25 min respectively, as compared with 7 h for Fructosamine 3-phosphates. NMR analysis indicated that 1-deoxy-1-morpholinopsicose (DMP, a substrate for FN3K and FN3K-RP), like 1-deoxy-1-morpholinofructose (DMF, a substrate of FN3K), penetrated erythrocytes and was converted into the corresponding 3-phospho-derivative. Incubation of erythrocytes with 50 mM allose, 200 mM glucose or 10 mM ribose for 24 h resulted in the accumulation of glycated haemoglobin, and this accumulation was approx. 1.9-2.6-fold higher if DMP, a competitive inhibitor of both FN3K and FN3K-RP, was present in the incubation medium. Incubation with 50 mM allose or 200 mM glucose also caused the accumulation of ketoamine 3-phosphates, which was inhibited by DMP. By contrast, DMF, a specific inhibitor of FN3K, only affected the glucose-dependent accumulation of glycated haemoglobin and ketoamine 3-phosphates. These data indicate that FN3K-RP can phosphorylate intracellular, protein-bound psicosamines and ribulosamines, thus leading to deglycation.
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a mammalian protein homologous to Fructosamine 3 kinase is a ketosamine 3 kinase acting on psicosamines and ribulosamines but not on Fructosamines
Diabetes, 2003Co-Authors: François Collard, Ghislain Delpierre, Vincent Stroobant, Gert Matthijs, Emile Van SchaftingenAbstract:Fructosamine-3-kinase (FN3K) is an enzyme that appears to be responsible for the removal of Fructosamines from proteins. In this study, we report the sequence of human and mouse cDNAs encoding proteins sharing 65% sequence identity with FN3K. The genes encoding FN3K and FN3K-related protein (FN3K-RP) are present next to each other on human chromosome 17q25, and they both have a similar 6-exon structure. Northern blots of mouse tissues RNAs indicate a high level of expression of both genes in bone marrow, brain, kidneys, and spleen. Human FN3K-RP was transfected in human embryonic kidney (HEK) cells, and the expressed protein was partially purified by chromatography on Blue Sepharose. Unlike FN3K, FN3K-RP did not phosphorylate fructoselysine, 1-deoxy-1-morpholino-fructose, or lysozyme glycated with glucose. In a more systematic screening for potential substrates for FN3K-RP, we found, however, that both enzymes phosphorylated ketosamines with a D-configuration in C3 (psicoselysine, 1-deoxy-1-morpholino-psicose, 1-deoxy-1-morpholino-ribulose, lysozyme glycated with allose-the C3 epimer of glucose, or with ribose). Tandem mass spectrometry and nuclear magnetic resonance analysis of the product of phosphorylation of 1-deoxy-1-morpholino-psicose by FN3K-RP indicated that this enzyme phosphorylates the third carbon of the sugar moiety. These results indicate that FN3K-RP is a ketosamine-3-kinase (ketosamine-3-kinase 2). This enzyme presumably plays a role in freeing proteins from ribulosamines or psicosamines, which might arise in a several step process, from the reaction of amines with glucose and/or glycolytic intermediates. This role is shared by Fructosamine-3-kinase (ketosamine-3-kinase 1), which has, in addition, the unique capacity to phosphorylate Fructosamines.
Juliette Fortpied - One of the best experts on this subject based on the ideXlab platform.
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many Fructosamine 3 kinase homologues in bacteria are ribulosamine erythrulosamine 3 kinases potentially involved in protein deglycation
FEBS Journal, 2007Co-Authors: Rita Gemayel, Didier Vertommen, Juliette Fortpied, Rim Rzem, Maria Veigadacunha, Emile Van SchaftingenAbstract:The purpose of this work was to identify the function of bacterial homologues of Fructosamine 3-kinase (FN3K), a mammalian enzyme responsible for the removal of Fructosamines from proteins. FN3K homologues were identified in approximately 200 (i.e. approximately 27%) of the sequenced bacterial genomes. In 11 of these genomes, from phylogenetically distant bacteria, the FN3K homologue was immediately preceded by a low-molecular-weight protein-tyrosine-phosphatase (LMW-PTP) homologue, which is therefore probably functionally related to the FN3K homologue. Five bacterial FN3K homologues (from Escherichia coli, Enterococcus faecium, Lactobacillus plantarum, Staphylococcus aureus and Thermus thermophilus) were overexpressed in E. coli, purified and their kinetic properties investigated. Four were ribulosamine/erythrulosamine 3-kinases acting best on free lysine and cadaverine derivatives, but not on ribulosamines bound to the alpha amino group of amino acids. They also phosphorylated protein-bound ribulosamines or erythrulosamines, but not protein-bound Fructosamines, therefore having properties similar to those of mammalian FN3K-related protein. The E. coli FN3K homologue (YniA) was inactive on all tested substrates. The LMW-PTP of T. thermophilus, which forms an operon with an FN3K homologue, and an LMW-PTP of S. aureus (PtpA) were overexpressed in E. coli, purified and shown to dephosphorylate not only protein tyrosine phosphates, but protein ribulosamine 5-phosphates as well as free ribuloselysine 5-phosphate and erythruloselysine 4-phosphate. These LMW-PTPs were devoid of ribulosamine 3-phosphatase activity. It is concluded that most bacterial FN3K homologues are ribulosamine/erythrulosamine 3-kinases. They may serve, in conjunction with a phosphatase, to deglycate products of glycation formed from ribose 5-phosphate or erythrose 4-phosphate.
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magnesium dependent phosphatase 1 is a protein Fructosamine 6 phosphatase potentially involved in glycation repair
Journal of Biological Chemistry, 2006Co-Authors: Juliette Fortpied, Didier Vertommen, Pushpa Maliekal, Emile Van SchaftingenAbstract:Fructosamine-3-kinase (FN3K) is a recently described protein-repair enzyme responsible for the removal of Fructosamines, which are the products of a spontaneous reaction of glucose with amines. We show here that, compared with glucose, glucose 6-phosphate (Glu-6-P) reacted 3-6-fold more rapidly with proteins and 8-fold more rapidly with N-alpha-t-Boc-lysine, being therefore a more significant intracellular glycating agent than glucose in skeletal muscle and heart. Fructosamine 6-phosphates, which result from the reaction of amines with Glu-6-P, were not substrates for FN3K. However, a phosphatase that dephosphorylates protein-bound Fructosamine 6-phosphates was found to be present in rat tissues. This enzyme was purified to near homogeneity from skeletal muscle and was identified as magnesium-dependent phosphatase-1 (MDP-1), an enzyme of the haloacid dehalogenase family with a putative protein-tyrosine phosphatase function. Human recombinant MDP-1 acted on protein-bound Fructosamine 6-phosphates with a catalytic efficiency >10-fold higher than those observed with its next best substrates (arabinose 5-phosphate and free fructoselysine 6-phosphate) and >100-fold higher than with protein-phosphotyrosine. It had no detectable activity on Fructosamine 3-phosphates. MDP-1 dephosphorylated up to approximately 75% of the Fructosamine 6-phosphates that are present on lysozyme after incubation of this protein with Glu-6-P. Furthermore, lysozyme glycated with Glu-6-P was converted by MDP-1 to a substrate for FN3K. We conclude that MDP-1 may act physiologically in conjunction with FN3K to free proteins from the glycation products derived from Glu-6-P.
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Fructosamine 3 kinase related protein and deglycation in human erythrocytes
Biochemical Journal, 2004Co-Authors: François Collard, Elsa Wiame, Ghislain Delpierre, Didier Vertommen, Juliette Fortpied, Niki Bergans, Florent Vanstapel, Emile Van SchaftingenAbstract:Fructosamine 3-kinase (FN3K), an enzyme initially identified in erythrocytes, catalyses the phosphorylation of Fructosamines on their third carbon, leading to their destabilization and their removal from protein. We show that human erythrocytes also contain FN3K-related protein (FN3K-RP), an enzyme that phosphorylates psicosamines and ribulosamines, but not Fructosamines, on the third carbon of their sugar moiety. Protein-bound psicosamine 3-phosphates and ribulosamine 3-phosphates are unstable, decomposing at pH 7.1 and 37 degrees C with half-lives of 8.8 h and 25 min respectively, as compared with 7 h for Fructosamine 3-phosphates. NMR analysis indicated that 1-deoxy-1-morpholinopsicose (DMP, a substrate for FN3K and FN3K-RP), like 1-deoxy-1-morpholinofructose (DMF, a substrate of FN3K), penetrated erythrocytes and was converted into the corresponding 3-phospho-derivative. Incubation of erythrocytes with 50 mM allose, 200 mM glucose or 10 mM ribose for 24 h resulted in the accumulation of glycated haemoglobin, and this accumulation was approx. 1.9-2.6-fold higher if DMP, a competitive inhibitor of both FN3K and FN3K-RP, was present in the incubation medium. Incubation with 50 mM allose or 200 mM glucose also caused the accumulation of ketoamine 3-phosphates, which was inhibited by DMP. By contrast, DMF, a specific inhibitor of FN3K, only affected the glucose-dependent accumulation of glycated haemoglobin and ketoamine 3-phosphates. These data indicate that FN3K-RP can phosphorylate intracellular, protein-bound psicosamines and ribulosamines, thus leading to deglycation.
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Fructosamine 3 kinase is involved in an intracellular deglycation pathway in human erythrocytes
Biochemical Journal, 2002Co-Authors: Ghislain Delpierre, François Collard, Juliette Fortpied, Emile Van SchaftingenAbstract:Fructosamine 3-kinase, which phosphorylates low-molecular-mass and protein-bound Fructosamines on the third carbon of their deoxyfructose moiety, is quite active in erythrocytes, and was proposed to initiate a process removing Fructosamine residues from proteins. In the present study, we show that incubation of human erythrocytes with 200 mM glucose not only caused the progressive formation of glycated haemoglobin, but also increased the level of an anionic form of haemoglobin containing alkali-labile phosphate, to approx. 5% of total haemoglobin. 1-Deoxy-1-morpholinofructose (DMF), a substrate and competitive inhibitor of Fructosamine 3-kinase, doubled the rate of accumulation of glycated haemoglobin, but markedly decreased the amount of haemoglobin containing alkali-labile phosphate. The latter corresponds therefore to haemoglobin bound to a Fructosamine 3-phosphate group (FN3P-Hb). Returning erythrocytes incubated with 200 mM glucose and DMF to a low-glucose medium devoid of DMF caused a decrease in the amount of glycated haemoglobin, a transient increase in FN3P-Hb and a net decrease in the sum (glycated haemoglobin+FN3P-Hb). These effects were prevented by DMF, indicating that Fructosamine 3-kinase is involved in the removal of Fructosamine residues. The second step of this 'deglycation' process is most likely a spontaneous decomposition of the Fructosamine 3-phosphate residues to a free amine, 3-deoxyglucosone and P(i). This is consistent with the findings that 2-oxo-3-deoxygluconate, the product of 3-deoxyglucosone oxidation, is formed in erythrocytes incubated for 2 days with 200 mM glucose in a sufficient amount to account for the removal of Fructosamine residues from proteins, and that DMF appears to inhibit the formation of 2-oxo-3-deoxygluconate from elevated glucose concentrations.
