Post-translational protein modifications in type 1 diabetes: a role for the repair enzyme protein-L-isoaspartate (D-aspartate) O-methyltransferase?

AIMS/HYPOTHESIS: Post-translational modifications, such as isomerisation of native proteins, may create new antigenic epitopes and play a role in the development of the autoimmune response. Protein-L-isoaspartate (D-aspartate) O-methyltransferase (PIMT), encoded by the gene PCMT1, is an enzyme that recognises and repairs isomerised Asn and Asp residues in proteins. The aim of this study was to assess the role of PIMT in the development of type 1 diabetes. MATERIALS AND METHODS: Immunohistochemical analysis of 59 normal human tissues was performed with a monoclonal PIMT antibody. CGP3466B, which induces expression of Pcmt1, was tested on MIN6 and INS1 cells, to assess its effect on Pcmt1 mRNA and PIMT levels (RT-PCR and western blot) and apoptosis. Forty-five diabetes-prone BioBreeding (BB) Ottawa Karlsburg (OK) rats were randomised to receive 0, 14 or 500 microg/kg (denoted as the control, low-dose and high-dose group, respectively) of CGP3466B from week 5 to week 20. RESULTS: A high level of PIMT protein was detected in beta cells. CGP3466B induced a two- to threefold increase in Pcmt1 mRNA levels and reduced apoptosis by 10% in MIN6 cells. No significant effect was seen on cytokine-induced apoptosis or PIMT protein levels in INS1 cells. The onset of diabetes in the BB/OK rats was significantly delayed (85.6+/-9.0 vs 84.3+/-6.8 vs 106.6+/-13.5 days, respectively; p<0.01 for high-dose vs low-dose and control groups), the severity of the disease was reduced (glucose 22.2+/-3.2 vs 16.9+/-2.6 vs 15.8+/-2.7 mmol; p<0.01 for high- and low-dose groups vs control group) and residual beta cells were more frequently identified (43% vs 71% vs 86%; p<0.05 for high-dose vs control group) in the treated animals. CONCLUSIONS/INTERPRETATION: The results support a role for post-translational modifications and PIMT in the development of type 1 diabetes in the diabetes-prone BB rat, and perhaps also in humans.

Effects of ibuprofen on molecular markers of cartilage and synovium turnover in patients with knee osteoarthritis.

OBJECTIVE: The aim of this study was to evaluate the effect of ibuprofen on the urinary excretion of C-terminal crosslinking telopeptide of type II collagen (CTX-II) and urinary glucosyl galactosyl pyridinoline (Glc-Gal-PYD), two new molecular markers of cartilage and synovial tissue metabolism, respectively, in patients with knee osteoarthritis (OA). METHODS: We studied 201 patients with knee pain and radiographic evidence of knee OA who were on treatment with non-steroidal anti-inflammatory drugs (NSAIDs) prior to study initiation. After an initial screening visit, patients were withdrawn from their pre-study NSAID and, following a flare of their OA symptoms, were randomised to ibuprofen (2400 mg/day) or placebo. Urinary CTX-II and Glc-Gal-PYD levels were measured at time of randomisation (baseline) and after 4-6 weeks of treatment. RESULTS: After 4 to 6 weeks, urinary CTX-II (+17%, p = 0.023) and Glc-Gal-PYD (+10%, p = 0.020) increased significantly from baseline in the placebo group whereas marginal or no increase was observed in the ibuprofen group (CTX-II +2%, NS and Glc-Gal-PYD +4%, p = 0.045). For urinary CTX-II, the difference in the change from baseline between placebo and ibuprofen treated groups was significant (13%, p = 0.017). At baseline, urinary levels of CTX-II and Glc-Gal-PYD were higher in patients with knee swelling (n = 127) than in those without (n = 74) (p<0.02 for both markers). When patients were stratified according to presence or absence of knee swelling at baseline, the increases over 4-6 weeks of urinary CTX-II and Glc-Gal-PYD in the placebo group were restricted to patients with knee swelling (+22% from baseline, p = 0.001 and +12%, p = 0.011, for urinary CTX-II and Glc-Gal-PYD respectively). In patients with knee swelling who were treated with ibuprofen this increase was not observed and the difference from placebo was significant for urinary CTX-II (p = 0.014). CONCLUSION: In patients with a flare of knee OA, specifically in patients with evidence of joint inflammation documented by knee swelling, there was a significant increase in markers reflecting cartilage and synovium metabolism that could partly be prevented by high doses of ibuprofen. These data suggest that patients with a flare of knee OA are characterised by increased cartilage and synovial tissue degradation, which may be partly prevented by high doses of NSAIDs.

Mechanism of circadian variation in bone resorption.

The diurnal variation in bone resorption markers is poorly understood and may contain essential information about regulation of bone resorption. To explore the acute regulation of bone resorption we studied bone turnover in 14 postmenopausal women during a randomized, crossover, 24 h study of oral glucose tolerance test (OGTT), normal diet, or fasting. Whereas fasting counteracted variation in bone resorption, as measured by serum C-telopeptide fragments of collagen type 1 degradation (s-CTx), OGTT and normal diet induced a 50% reduction (p < 0.001) over 2 h. For OGTT, s-CTx reverted to baseline levels after 6 h, and for normal diet s-CTx remained suppressed during the afternoon and returned to baseline overnight. Repeated OGTT at 8:00 A.M. and 8:00 P.M. in nine postmenopausal women demonstrated that identical reductions in s-CTx could be obtained at both timepoints with an intermediate return to baseline between tests. A 2 h randomized, crossover study of OGTT and fasting in 23 men and premenopausal women similarly revealed a 50% decrease in s-CTx. A randomized, crossover 2 h study of insulin tolerance test compared with fasting in six men and premenopausal women demonstrated a 20%-30% decrease in s-CTx (p < 0.01-0.05). Nine hour follow-up of ten healthy individuals during a crossover experiment of OGTT, protein, and fat intake revealed a comparable 50% reduction in s-CTx, but distinct profiles of serum glucose and serum insulin. Bone resorption was reduced by intake of food, glucose, fat, and protein and counteracted by fasting, and this seems to have been be independent of age and gender. Both exogenous and endogenous insulin stimulation tests induced a reduction in bone resorption, but this was only partial when compared with the reduction observed during food intake.

Peptide amidation by enzymatic transacylation and photolysis.

A series of model peptides with a C-terminal protected amide group were prepared by enzymatic transacylation. The protection groups were removed by photolysis to give the warranted peptide amides in high yields. Furthermore, fragments of human calcitonin were prepared. Various protective groups were employed, and the pH, solvent and concentration dependency of the enzymatic transcylation were examined. The photo-cleavage reaction was examined for wavelength, concentration and pH dependency. It was shown that the optimal yields required addition of a chemical scavenger for the photolysis byproducts.

C-terminal amidation of calcitonin by carboxypeptidase Y catalyzed transpeptidation with a photocleavable nucleophile.

The C-terminal amidation of calcitonin represents an important technological problem. A method using a serine carboxypeptidase-catalyzed transpeptidation reaction in combination with photochemical cleavage to give the warranted peptide amide is described. The overall yield is higher than 95%.