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 plasminogen activator accumulated 0.77 mol of isoaspartate per mol of plasminogen activator over a 14-day period. Isoaspartate was detected by enzymatic transfer of 3H-labeled methyl groups from S-adenosyl-L-methionine in a reaction catalyzed by protein L-isoaspartyl methyltransferase. Analysis of tryptic peptides derived from aged plasminogen activator revealed that the two major sites of isoaspartate accumulation resulted from deamidation of Asn58 in the sequence -FNGG- and Asn177 in the sequence -GNSD-. Significant levels of isoaspartate also accumulated via deamidation of Asn37 in the sequence -CNSG-. All three sites occur in sequences predicted from studies with synthetic peptide to be unstable. All three sites appear to be on the surface of the protein, and all three occur in regions of the protein predicted to have higher than average chain mobility. These findings add support to the idea that sequence and flexibility play major roles in determining susceptibility to deamidation and peptide bond isomerization at Asn and Asp sites under mild conditions. These studies also illustrate the utility of enzymatic methylation for characterizing sites of deamidation in a large protein that contains numerous disulfide bonds and several sites of glycosylation.
Paranandi
M V
MV
Department of Molecular Biology and Biochemistry, University of California, Irvine 92717-3900.
Guzzetta
A W
AW
Hancock
W S
WS
Aswad
D W
DW
D000478:Alkylation; D000577:Amides / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid / Q000378:metabolism*; D016466:CHO Cells; D006224:Cricetinae; D004220:Disulfides / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008745:Methylation; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010449:Peptide Mapping; D011994:Recombinant Proteins / Q000378:metabolism; D010959:Tissue Plasminogen Activator / Q000378:metabolism*; D014357:Trypsin / Q000378:metabolism
D000577:Amides; D004220:Disulfides; D011994:Recombinant Proteins; D001224:Aspartic Acid; D014357:Trypsin; D010959:Tissue Plasminogen Activator