Mutants of human insulin-like growth factor II (IGF II). Expression and characterization of truncated IGF II and of two naturally occurring variants.
Insulin-like growth factor II (IGF II) and four structural analogs, constructed by site-directed mutagenesis, were expressed as protein A fusion proteins in Escherichia coli BL21pLysS cells, cleaved with cyanogen bromide and purified by affinity chromatography and HPLC. Two mutants (Ser29 substituted by Arg-Leu-Pro-Gly, and Ser33 substituted by Cys-Gly-Asp) represent two naturally occurring variants of IGF II. The other two mutants, (7-67)IGF II and (9-67)IGF II, are truncated at the amino-terminus in analogy to the naturally occurring des(1-3)IGF I ('truncated IGF I'). These mutants were tested for their binding affinities to type-1 and type-2 IGF receptors, to IGF binding protein-3 (IGFBP-3) and for their stimulation of thymidine incorporation into DNA. The affinities of the Ser29 and Ser33 mutants to the type-1 IGF receptor were 85% and 39%, respectively, compared to wild-type IGF II, those of (7-67)IGF II and (9-67)IGF II 96% and 15%, respectively. The potencies of the Ser33 and the (9-67) mutant to stimulate thymidine incorporation into DNA correlated closely with the affinities to the type-1 IGF receptor, whereas the bioavailability of the Ser29 mutant was lower and that of the (7-67) mutant higher than the type-1 receptor binding, possibly due to interferences with endogenously secreted IGFBPs. The affinities of the Ser29 and Ser33 mutants to the type-2 IGF receptor were 110% and 71%, respectively, those of the two truncated mutants 25% and 23%, respectively. The affinity of the Ser29 mutant to IGFBP-3 was increased to 171%, whereas those of the Ser33 mutant and the two truncated mutants were reduced (34%, 10% and 19%, respectively).
Lüthi
C
C
Department of Biochemistry, University of Zürich, Switzerland.
Roth
B V
BV
Humbel
R E
RE
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D002352:Carrier Proteins / Q000378:metabolism; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004261:DNA Replication / Q000187:drug effects; D006801:Humans; D018969:Insulin-Like Growth Factor Binding Proteins; D007335:Insulin-Like Growth Factor II / Q000235:genetics* / Q000378:metabolism* / Q000494:pharmacology; D007700:Kinetics; D008360:Mannosephosphates / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D017527:Receptor, IGF Type 2; D011956:Receptors, Cell Surface / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism / Q000494:pharmacology; D015183:Restriction Mapping; D013936:Thymidine / Q000378:metabolism
D002352:Carrier Proteins; D018969:Insulin-Like Growth Factor Binding Proteins; D008360:Mannosephosphates; D009838:Oligodeoxyribonucleotides; D017527:Receptor, IGF Type 2; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; D007335:Insulin-Like Growth Factor II; D004247:DNA; D013936:Thymidine