HUMAN CILIARY NEUROTROPHIC FACTOR - A STRUCTURE-FUNCTION ANALYSIS

Ciliary neurotrophic factor (CNTF) promotes survival in vitro and in v ivo of several neuronal cell types including sensory and motor neurons . The primary structure of CNTF suggests it to be a cytosolic protein with strong similarity to the alpha-helical cytokine family which is c haracterized by a bundle of four anti-parallel helices. CNTF exerts it s activity via complexation with CNTF receptor (CNTF-R). This complex consists of a CNTF-binding protein (CNTF-R) and two proteins important for signal transduction [gp130 and leukaemia inhibitory factor recept or (LIF-R)]. We have shortened the cDNA coding for CNTF at both the 5' and the 3' end and expressed the truncated proteins in bacteria. Biol ogical activities of the protein preparations were determined by their ability to induce proliferation of BAF/3 cells that were stably trans fected with CNTF-R, gp130 and LIF-R cDNAs. CNTF proteins with 14 amino acid residues removed from the N-terminus were biologically active wh ereas the removal of 23 amino acids resulted in an inactive protein. I n addition, 18 amino acid residues could be removed from the C-terminu s of the CNTF protein without apparent loss of bioactivity, but furthe r truncation at the C-terminus yielded biologically inactive proteins. The introduction of two point mutations into the CNTF protein at a si te that presumably interacts with one of the two signal-transducing pr oteins resulted in a CNTF mutant with no measurable bioactivity. In ad dition, a model of the three-dimensional structure of human CNTF was c onstructed using the recently established structural co-ordinates of t he related cytokine, granulocyte colony-stimulating factor. CD spectra of CNTF together with our mutational analysis and our three-dimension al model fully support the view that CNTF belongs to the family of alp ha-helical cytokines. It is expected that our results will facilitate the rational design of CNTF mutants with agonistic or antagonistic pro perties.