Increased protein glycation in fructosamine 3-kinase-deficient mice

Abstract

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.

Figure 1. Targeted deletion in the FN3K locus in mice

(A) The hatched boxes represent the exons in the Fn3k gene, the grey boxes those in the Fn3k-RP gene [34] and the white boxes the neomycin-resistant cassette (Neo) and the β-galactosidase marker (β-Gal). Key restriction enzyme sites and localization of probes used during Southern blotting are shown under the disrupted allele. (B) Confirmation of the correct targeting of the 5′ and 3′ arms and excludes any additional insertion by Southern blotting. WT, wild-type; KO, knock-out. (C) Confirmation that transcription from the Fn3k locus has been ablated in various tissues in mice that are homozygous for the Fn3k deletion. RT-PCR analysis of the sequence spanning exons 1 (forward primer: 5′-CGTGTTTGTCAAGGTCAATCG-3′) to 3 (reverse primer: 5′-TGCCATCTGTTCCCCGAGC-3′) shows the absence of a detectable transcript in Fn3k^−/− mice. (D) FN3K activity in erythrocytes, kidney and brain following the ablation of the Fn3k gene in mouse. The activity of FN3K is related to the amount of protein in the extract. In erythrocytes the activity was determined in extracts of two mice for each genotype. In kidney and brain extracts, the activity in each tissue was determined after pooling the extracts from two mice of each genotype prior to purification.

Figure 2. Glycation of haemoglobin in wild-type and Fn3k^−/− mice

Total GlcHb that binds to phenylboronate affinity columns in erythrocytes from wild-type Fn3k^+/+, Fn3k^+/− and Fn3k^−/− male (A) and female (B) mice aged 1, 2, 4 and 6 months. Each point represents the mean±S.E.M. from four mice.

Figure 3. Phosphorylation of glycated residues from proteins in erythrocyte extracts from wild-type and Fn3k^−/− mice

Whole blood extracts and erythrocyte lysates from Fn3k^+/+ and Fn3k^−/− mice (100 μg per lane) or control lysozyme or glycated (25 μg per lane) were separated by SDS/PAGE, the proteins were transferred on to a nitrocellulose membrane and phosphorylated with recombinant E. coli fructoselysine 6-kinase and [γ-³²P]ATP. The membrane was overloaded to enhance the signal of the faintest bands. As a result of this, the phosphorylation of the lower band, corresponding to haemoglobin monomers, is underestimated compared with the other bands.

Figure 4. HPLC profile of radioactive peptides after tryptic digestion of haemoglobin from Fn3k^+/+ and Fn3k^−/− mice phosphorylated with FN3K and [γ-³²P]ATP

Haemoglobin from Fn3k^+/+ and Fn3k^−/− mice was partially purified on DEAE-Sepharose and incubated with recombinant mouse FN3K and [γ-³²P]ATP for 2 h in vitro. After reduction of fructosamine 3-[³²P]phosphate with NaBH₄, protein was precipitated in acid, resuspended in urea and digested overnight with trypsin [13]. Peptides were separated by reverse-phase HPLC and radioactive peaks were detected by Cerenkov counting.

Figure 5. Glycation of cytosolic enzymes (TPI, PGI and 3-PGK) in erythrocytes from wild-type and Fn3k^−/− mice

The glycation in (A) TPI, (B) PGI and (C) 3-PGK was measured in extracts from erythrocytes from wild-type and Fn3k^−/− mice aged 1, 2, 4 and 6 months after separation of the glycated and the non-glycated forms of the enzymes by phenylboronate affinity chromatography as described in the Experimental section. Error bars are calculated from measurements in two animals.

Figure 6. Increase in intracellular glycation of TPI, PGI and 3-PGK from liver, kidney, brain and muscle of FN3K-deficient mice

Tissue extracts were diluted in the lysis solution and BSA was added to standardize the protein concentration to 2 mg in 100 μl, which was then loaded onto the phenylboronate affinity columns. Non-glycated and glycated proteins were separated as described in the legend for Figure 5. The results are means±S.E.M. of four measurements for extracts from mice aged 1, 2, 4 and 6 months. The difference between the values obtained for the samples from the wild-type and Fn3k^−/− mice were compared using a Student's t test (***P<0.001; **0.001≤P<0.01; and, *0.01≤P<0.05).