Exchange of a single amino acid interconverts the specific activity and gel mobility of human and rat ciliary neurotrophic factors.
Human and rat ciliary neurotrophic factors (CNTF), which share 85% sequence identity, promote the survival of chicken embryo ciliary ganglia neurons in vitro, but display a 4-5-fold difference in specific activity. To explore the origin of this difference and gain insight into the structural organization of CNTF, we created chimeric proteins of these two species. Surprisingly, we found that the differences in two apparently unrelated properties, gel mobility and specific activity, resided in a single amino acid. Substituting arginine residue 63 of rat CNTF into the human sequence created a protein with the properties of rat CNTF. Conversely, substituting the human CNTF glutamine residue 63 into rat CNTF generated a protein with the properties of human CNTF. Binding experiments confirmed that the distinct specific activities of human and rat CNTF and their chimeras reside in structural differences among these ligands rather than species differences in their receptors. Alanine substitution (Q63A) had no effect on the properties of human CNTF, whereas the R63A substitution reduced both the gel mobility and the specific activity of rat CNTF. Finally, a Q95R substitution at a different position of human CNTF had no effect on its properties. These results demonstrate that Arg-63 is both specific and critical in determining the structural differences of human and rat CNTF.
REGENERON Pharmaceuticals Inc., Tarrytown, New York 10591-6707.
N
N
Panayotatos
E
E
Radziejewska
A
A
Acheson
D
D
Pearsall
A
A
Thadani
V
V
Wong
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D002642:Chick Embryo; D020934:Ciliary Neurotrophic Factor; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008969:Molecular Sequence Data; D009414:Nerve Growth Factors / Q000737:chemistry* / Q000235:genetics; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016133:Polymerase Chain Reaction; D015202:Protein Engineering; D017433:Protein Structure, Secondary; D051381:Rats; D020801:Receptor, Ciliary Neurotrophic Factor; D011956:Receptors, Cell Surface / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D013329:Structure-Activity Relationship
D020934:Ciliary Neurotrophic Factor; D009414:Nerve Growth Factors; D009419:Nerve Tissue Proteins; D020801:Receptor, Ciliary Neurotrophic Factor; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins