DEAMIDATION AND ISOASPARTATE FORMATION DURING IN-VITRO AGING OF RECOMBINANT TISSUE-PLASMINOGEN ACTIVATOR

When incubated at pH 7.3, 37-degrees-C, human recombinant tissue plasm inogen activator accumulated 0.77 mol of isoaspartate per mol of plasm inogen activator over a 14-day period. Isoaspartate was detected by en zymatic transfer of H-3-labeled methyl groups from S-adenosyl-L-methio nine in a reaction catalyzed by protein L-isoaspartyl methyltransferas e. Analysis of tryptic peptides derived from aged plasminogen activato r revealed that the two major sites of isoaspartate accumulation resul ted from deamidation of Asn58 in the sequence -FNGG- and Asn77 in the sequence -GNSD-. Significant levels of isoaspartate also accumulated v ia deamidation of Asn37 in the sequence -CNSG-. All three sites occur in sequences predicted from studies with synthetic peptide to be unsta ble. All three sites appear to be on the surface of the protein, and a ll three occur in regions of the protein predicted to have higher than average chain mobility. These findings add support to the idea that s equence and flexibility play major roles in determining susceptibility to deamidation and peptide bond isomerization at Asn and Asp sites un der mild conditions. These studies also illustrate the utility of enzy matic methylation for characterizing sites of deamidation in a large p rotein that contains numerous disulfide bonds and several sites of gly cosylation.