On the structure of platelet-derived growth factor AA: C-terminal processing, epitopes, and characterization of cysteine residues.
The complete amino acid sequence analysis of the "short" form of rPDGF-AA expressed in baby hamster kidney cells revealed the absence of posttranslationally modified amino acid. Approximately 50% of the proteins were shortened by two to three amino acid residues at the C-terminus. Trypsin treatment of BHK rPDGF-AA lead to the identification of two internal epitopes that correspond to the two previously described domains in rPDGF-BB [Vogel, S., & Hoppe, J. (1989) Biochemistry 28, 2961-2966]. Cysteine residues at positions 37, 46, 47, and 93, respectively, were converted by site-directed mutagenesis into serine residues, and the monomeric proteins were prepared through expression in Escherichia coli. None of the mutant proteins was able to dimerize, but all of them exhibited to various extents a reversible conformational change which may reflect an intermediate prefolded monomer. An intramolecular disulfide bridge between Cys-10 and Cys-91/93 was identified in these monomers. From a mixture of the mutant proteins 37 and 46, an active dimer was reconstituted, suggesting an intermolecular cysteine bridge between these two residues.
Jaumann
M
M
Physiologisch-Chemisches Institut, Universität Würzburg, Biozentrum, FRG.
Hoppe
V
V
Tatje
D
D
Eichner
W
W
Hoppe
J
J
D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine; D004220:Disulfides / Q000032:analysis; D000939:Epitopes / Q000032:analysis*; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000302:isolation & purification; D010982:Platelet-Derived Growth Factor / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000494:pharmacology; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology / Q000494:pharmacology; D014357:Trypsin
D004220:Disulfides; D000939:Epitopes; D046911:Macromolecular Substances; D010446:Peptide Fragments; D010982:Platelet-Derived Growth Factor; D011994:Recombinant Proteins; D014357:Trypsin; D003545:Cysteine