Advanced glycation end-products (AGEs) form at an accelerated rate in diabetes and are associated with the development of diabetic complications. So far, the effect of AGEs on proteases was mostly investigated with substrates modified non-specifically with hexoses amongst others in combination with proteases of broad specificity. Here, we investigated the effect of AGEs on the proinsulin processing peptidases preprotein convertase 1 (PC1) and carboxypeptidase E (CPE) via two ways: - Measuring of activity towards peptide substrates. These contained chemically defined AGEs carboxymethyllysine (CML) or methylglyoxyl-hydroimidazolone-1 (MGH1) or unmodified amino acids. - Recombinant human proinsulin was incubated with methylglyoxal for 24 h and was processed by addition of PC1 and CPE in vitro. Formation of insulin was compared to unmodified proinsulin. In parallel formation of AGE-modified proinsulin was quantified.
Artificial peptide substrates were synthesized by solid phase peptide synthesis and purified by HPLC. A protected building block was used for MGH1 incorporation while CML was introduced on the resin. Protease activity towards artificial substrates was measured by fluorescence assay or LC MS/MS. Proinsulin, insulin and AGE-modified proinsulin were quantified by LC MS.
Strong activity of PC1 and CPE was seen towards arginine containing substrates. Activity was completely absent towards modified amino acids MGH1 and CML for both, PC1 and CPE. Incubation of proinsulin with MG resulted in MGH1 modified proinsulin. At low MG (µM) concentrations this was associated with a 25 % reduction of insulin formation while high MG concentrations (95 µM) led to a 75 % reduction in insulin formation.
Here we demonstrate for the first time the effect of substrate modifications with AGEs on physiologically important proteases PC1 and CPE. The latter are not only involved in the processing of proinsulin but a range of endocrinologically and neurophysiologically important peptide hormones.
W. Mier: None. B. Beijer: None. J.M. Szendroedi: Consultant; Boehringer Ingelheim International GmbH. T.H. Fleming: None.
DFG (SFB 1118)