Maria Veiga-da-Cunha

Sequence of a putative glucose 6-phosphate translocase, mutated in glycogen storage disease type Ib.

We report the sequence of a human cDNA that encodes a 46 kDa transmembrane protein homologous to bacterial transporters for phosphate esters. This protein presents at its carboxy terminus the consensus motif for retention in the endoplasmic reticulum. Northern blots of rat tissues indicate that the corresponding mRNA is mostly expressed in liver and kidney. In two patients with glycogen storage disease type Ib, mutations were observed that either replaced a conserved Gly to Cys or introduced a premature stop codon. The encoded protein is therefore most likely the glucose 6‐phosphate translocase that is functionally associated with glucose‐6‐phosphatase.

Human l‐3‐phosphoserine phosphatase: sequence, expression and evidence for a phosphoenzyme intermediate

We report the sequence of the cDNA encoding human l‐3‐phosphoserine phosphatase. The encoded polypeptide contains 225 residues and shows 30% sequence identity with the Escherichia coli enzyme. The human protein was expressed in a bacterial expression system and purified. Similar to known l‐3‐phosphoserine phosphatases, it catalyzed the Mg2+‐dependent hydrolysis of l‐phosphoserine and an exchange reaction between l‐serine and l‐phosphoserine. In addition we found that the enzyme was phosphorylated upon incubation with l‐[32P]phosphoserine, which indicates that the reaction mechanism proceeds via the formation of a phosphoryl‐enzyme intermediate. The sensitivity of the phosphoryl‐enzyme to alkali and to hydroxylamine suggests that an aspartyl‐ or a glutamyl‐phosphate was formed. The nucleotide sequence of the cDNA described in this article has been deposited in the EMBL data base under accession number Y10275.

Variability in erythrocyte fructosamine 3-kinase activity in humans correlates with polymorphisms in the FN3K gene and impacts on haemoglobin glycation at specific sites.

BACKGROUND Part of the fructosamines that are bound to intracellular proteins are repaired by fructosamine 3-kinase (FN3K). Because subject-to-subject variations in erythrocyte FN3K activity could affect the level of glycated haemoglobin independently of differences in blood glucose level, we explored if such variability existed, if it was genetically determined by the FN3K locus on 17q25 and if the FN3K activity correlated inversely with the level of glycated haemoglobin. RESULTS The mean erythrocyte FN3K activity did not differ between normoglycaemic subjects (n = 26) and type 1 diabetic patients (n = 31), but there was a wide interindividual variability in both groups (from about 1 to 4 mU/g haemoglobin). This variability was stable with time and associated (P < 0.0001) with two single nucleotide polymorphisms in the promoter region and exon 6 of the FN3K gene. There was no significant correlation between FN3K activity and the levels of HbA1c, total glycated haemoglobin (GHb) and haemoglobin fructoselysine residues, either in the normoglycaemic or diabetic group. However, detailed analysis of the glycation level at various sites in haemoglobin indicated that the glycation level of Lys-B-144 was about twice as high in normoglycaemic subjects with the lowest FN3K activities as compared to those with the highest FN3K activities. CONCLUSION Interindividual variability of FN3K activity is substantial and impacts on the glycation level at specific sites of haemoglobin, but does not detectably affect the level of HbA1c or GHb. As FN3K opposes one of the chemical effects of hyperglycaemia, it would be of interest to test whether hypoactivity of this enzyme favours the development of diabetic complications.

Mutations responsible for 3-phosphoserine phosphatase deficiency

We report the identification of the mutations in the only known case of L-3-phosphoserine phosphatase deficiency, a recessively inherited condition. The two mutations correspond to the replacement of the semiconserved Asp32 residue by an asparagine and of the extremely conserved Met52 by a threonine. The effects of both mutations were studied on the human recombinant enzyme, expressed in Escherichia coli. Met52Thr almost abolished the enzymatic activity, whereas the Asp32Asn mutation caused a 50% decrease in Vmax. In addition, L-serine, which inhibits the conversion of [14C] phosphoserine to serine when catalysed by the wild-type enzyme, had a lesser inhibitory effect on the Asp32Asn mutant, indicating a reduction in the rate of phosphoenzyme hydrolysis. These modifications in the properties of the enzyme are consistent with the modification in the kinetic properties observed in fibroblasts from the patient.

Mutations in the glucokinase regulatory protein gene in 2p23 in obese French caucasians.

Aims/HypothesisGlucokinase regulatory protein (GKRP) controls the activity of glucokinase in liver but possibly also in some areas of the central nervous system, suggesting that it could play a role in body mass control. Its gene is located in a region (2p21–23) linked to serum leptin levels. Our goal was to investigate whether mutations in the GKRP gene were associated with obesity.MethodsMutations were sought in the GKRP gene of 57 patients from the families of the French genome-wide scan for obesity that contributed most to the positive LOD score with 2p21–23. The identified mutations were further sought in 720 unrelated obese individuals and 384 individuals of normal weight and their effect on the properties of recombinant GKRP were investigated.ResultsThe most frequent mutation (Pro446Leu) had a similar allele frequency in the obese (0.63) and normal weight (0.64) subjects and did not affect the properties of GKRP. Similarly, no effect on the properties of GKRP was observed with Arg590Tyr, found in 10 out of 720 obese subjects and in 2 out of 384 control subjects (p=0.18). Mutation Arg227Stop was found in one obese family and in 1 out of 384 control subjects and led to an insoluble protein. Mutation Arg518Gln, replacing a conserved residue, led to a marked decrease in the affinity of GKRP for both fructose 6-phosphate and fructose 1-phosphate and to a destabilization of GKRP. However, this mutation did not co-segregate with obesity in the single family in which it was found.Conclusions/interpretationMutations that affect the properties of GKRP are found in the French population, but they do not seem to account for the linkage between the 2p23 locus and quantitative markers of obesity.

Effects of fructosamine-3-kinase deficiency on function and survival of mouse pancreatic islets after prolonged culture in high glucose or ribose concentrations

Due to their high glucose permeability, insulin-secreting pancreatic beta-cells likely undergo strong intracellular protein glycation at high glucose concentrations. They may, however, be partly protected from the glucotoxic alterations of their survival and function by fructosamine-3-kinase (FN3K), a ubiquitous enzyme that initiates deglycation of intracellular proteins. To test that hypothesis, we cultured pancreatic islets from Fn3k-knockout (Fn3k(-/-)) mice and their wild-type (WT) littermates for 1-3 wk in the presence of 10 or 30 mmol/l glucose (G10 or G30, respectively) and measured protein glycation, apoptosis, preproinsulin gene expression, and Ca(2+) and insulin secretory responses to acute glucose stimulation. The more potent glycating agent d-ribose (25 mmol/l) was used as positive control for protein glycation. In WT islets, a 1-wk culture in G30 significantly increased the amount of soluble intracellular protein-bound fructose-epsilon-lysines and the glucose sensitivity of beta-cells for changes in Ca(2+) and insulin secretion, whereas it decreased the islet insulin content. After 3 wk, culture in G30 also strongly decreased beta-cell glucose responsiveness and preproinsulin mRNA levels, whereas it increased islet cell apoptosis. Although protein-bound fructose-epsilon-lysines were more abundant in Fn3k(-/-) vs. WT islets, islet cell survival and function and their glucotoxic alterations were almost identical in both types of islets, except for a lower level of apoptosis in Fn3k(-/-) islets cultured for 3 wk in G30. In comparison, d-ribose (1 wk) similarly decreased preproinsulin expression and beta-cell glucose responsiveness in both types of islets, whereas it increased apoptosis to a larger extent in Fn3k(-/-) vs. WT islets. We conclude that, despite its ability to reduce the glycation of intracellular islet proteins, FN3K is neither required for the maintenance of beta-cell survival and function under control conditions nor involved in protection against beta-cell glucotoxicity. The latter, therefore, occurs independently from the associated increase in the level of intracellular fructose-epsilon-lysines.

CHAPTER 6:The Biochemistry of Enzymes Producing Carnosine and Anserine

In vertebrates, carnosine synthase (EC 6.3.2.11) and carnosine N-methyltransferase (EC 2.1.1.22) are the intracellular enzymes that catalyze the formation of carnosine (β-alanyl-L-histidine) and its natural methylated derivative – anserine (β-alanyl-N-π-methyl-L-histidine), respectively. Much effort has been devoted to the elucidation of the physiological roles of these “enigmatic” imidazole dipeptides despite the still limited information available on the enzymes responsible for their biosynthesis. Carnosine synthase and chicken carnosine N-methyltransferase have only recently been molecularly identified as ATP-grasp domain containing protein 1 (ATPGD1) and histamine N-methyltransferase-like protein (HNMT-like), respectively, giving new insights into the biosynthesis of carnosine and anserine. These findings open new opportunities of research in the quest for the biological functions of imidazole dipeptides and in the assessment of their therapeutic potential. The aim of this chapter is to summarize the recent advances in our knowledge on carnosine- and anserine-producing enzymes.