Formation of isoaspartate in molecules containing NGR, DGR, or RGD motives and schematic representation of the potential effects on receptor binding. (A) NGR transition to isoDGR in conjugates and in natural proteins (eg, fibronectin) can occur by nucleophilic attack of the backbone NH center on the Asn side-chain amide carbonyl, leading to formation of a succinimide intermediate. Hydrolysis of succinimide leads to formation of isoDGR and DGR mixtures, with changes in charge and peptide bond length. Isoaspartate formation can occur, in principle, also at DGR sites in proteins, by aspartate isomerization, although with slower kinetics. These nonenzymatic reactions are thermodynamically spontaneous and the kinetics depend on surrounding sequences and microenvironmental factors. (B) Potential effect of isoaspartate formation in molecules containing NGR, RGD, or DGR on the interaction with endothelial cell membrane receptors. NGR-containing conjugates (drugs, particles, and viruses) can interact with an endothelial form of aminopeptidase N (CD13). Whether NGR sites of certain natural proteins (eg, fibronectin) can also interact with CD13 is unknown. Nonenzymatic formation of isoaspartate at NGR or RGD sites in fibronectin, and potentially in other proteins with low turnover, can generate isoDGR or RGisoD.⁴  While isoDGR and RGD can interact with integrins on the cells surface (eg, αvβ3), DGR and RGisoD are inactive. Protein-L-isoAsp-O-methyltransferase (PIMT) can enzymatically convert isoDGR to DGR and RGisoD to RGD. Thus, while PIMT may rescue RGD from RGisoD (eg, in aged fibronectin and collagen),^4,74,76  this enzyme may regulate in a negative manner the function of isoDGR. NGR sites may work, therefore, as molecular timers for the formation of new integrin binding sites with potential enzymatic regulation. In principle, similar reactions could occur also in proteins containing DGR.