Replacement of a labile aspartyl residue increases the stability of human epidermal growth factor.
Long-term storage of recombinant human epidermal growth factor (EGF), an important promoter of cell division, results in its conversion to a new species that elutes later than native EGF on a reverse-phase column. This new species, called EGF-X, has only 20% of the biological activity of native EGF. Peptide mapping indicated that the primary structure of EGF-X differs from that of native EGF solely within the first 13 residues. N-Terminal sequencing of EGF-X revealed that about 30% of the polypeptides have been cleaved at the Asp-3/Ser-4 bond. In addition, the yields after the His residue at position 10 were extremely low, indicating that a chemical modification occurs at residue 11 that is incompatible with Edman degradation. We hypothesized that aspartic acid 11 had been converted to an isoaspartyl residue, and this was confirmed with L-isoaspartyl/D-aspartyl methyltransferase, an enzyme that methylates the side-chain carboxyl group of L-isoaspartyl residues but does not recognize normal L-aspartyl residues. EGF-X, but not EGF, was found to be a substrate of this enzyme, and proteolytic digestion of EGF-X with thermolysin localized the site of methylation to a nine-residue peptide containing position 11. We did not observe formation of the isoaspartyl derivative in EGF that had been denatured by reduction of its disulfide bonds. In addition, replacement of the aspartyl residue at position 11 with glutamic acid resulted in a fully active EGF derivative that does not form detectable amounts of EGF-X. We propose that conversion of this aspartyl residue to isoaspartate is a significant nonenzymatic degradation reaction affecting this growth factor.(ABSTRACT TRUNCATED AT 250 WORDS)
George-Nascimento
C
C
Chiron Research Laboratories, Chiron Corporation, Emeryville, California 94608.
Lowenson
J
J
Borissenko
M
M
Calderón
M
M
Medina-Selby
A
A
Kuo
J
J
Clarke
S
S
Randolph
A
A
D000595:Amino Acid Sequence; D001224:Aspartic Acid; D002478:Cells, Cultured; D002918:Chymotrypsin; D004261:DNA Replication / Q000187:drug effects; D004355:Drug Stability; D004815:Epidermal Growth Factor / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology; D006801:Humans; D008745:Methylation; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000138:chemical synthesis; D010449:Peptide Mapping; D011994:Recombinant Proteins / Q000737:chemistry / Q000494:pharmacology; D013820:Thermolysin; D013936:Thymidine / Q000378:metabolism
D009842:Oligopeptides; D011994:Recombinant Proteins; D001224:Aspartic Acid; D004815:Epidermal Growth Factor; D002918:Chymotrypsin; D013820:Thermolysin; D013936:Thymidine