Dl cap region involved in the receptor recognition and neural cell survival activity of human ciliary neurotrophic factor (mutagenesis/cytokine/glycoprotein gpl30/antagonist)

Human ciliary neurotrophic factor (hCNTF), which promotes the cell survival and differentiation of motor and other neurons, is a protein belonging structurally to the a-helical cytokine family. hCNTF was subjected to threedimensional structure modeling and site-directed mutagenesis to analyze its structure-function relationship. The replacement of Lys-155 with any other amino acid residue resulted in abolishment of neural cell survival activity, and some of the Glu-153 mutant proteins had 5to 10-fold higher biological activity. The Dl cap region (around the boundary between the CD loop and helix D) of hCNTF, including both Glu-153 and Lys-155, was shown to play a key role in the biological activity of hCNTF as one of the putative receptor-recognition sites. In this article, the Dl cap region of the 4-helix-bundle proteins is proposed to be important in receptor recognition and biological activity common to a-helical cytokine proteins reactive with gpl3O, a component protein of the receptors. Ciliary neurotrophic factor (CNTF), originally described as a trophic factor supporting the survival of ciliary ganglion neurons in vitro (1), has been well known to promote the survival and differentiation of motor neurons and other neuronal cells in vitro and in vivo (2-4). The effectiveness of treatment with CNTF in motor-neuron-degenerative animal models such as wobbler (5) and pmn mice (6) and in the facial-nerve axotomy model (7) led to a clinical trial targeted to patients suffering from the neuronal degenerative disease amyotrophic lateral sclerosis (8, 9). In spite of the detailed analysis and accumulated information on biological activity and efficacy, little is yet known about the tertiary structures, the structure-function relationship of CNTF, or the mode of interaction with its receptors. CNTF functionally acts on neuronal cells as do neurotrophins (NTs) such as NT-3, NT-4/5, nerve growth factor, and brain-derived neurotrophic factor. However, it is greatly different from NTs in both the molecular structure and the mode of receptor interaction. Human CNTF (hCNTF) is a 200amino-acid-residue protein rich in a-helical structure, having 53% a-helices and 9% (3-turns (10). A secondary structure analysis suggested that CNTF had a structural topology similar to the a-helical cytokines, though the amino acid sequence of hCNTF had no homology with them. All a-helical cytokines such as growth hormone (GH), prolactin (PRL), erythropoietin (EPO), granulocyte colony-stimulating factor (G-CSF), leukemia inhibitory factor (LIF), and some interleukins (ILs) (11) have a common structure with a characteristic bundle of four antiparallel helices, A, B, C, and D. As for the receptor, CNTF binds to CNTF receptors (CNTFRs) consisting of CNTFR a, LIF receptor (LIFR), and gp13O, the last of which is a common glycoprotein component among the receptors for CNTF, interleukin 6 (IL-6), LIF, oncostatin M, and interleukin 11 (IL-11) (12-14). Both the AB loop region (amino acid residues between helix A and B) and helix D have been proposed so far to be important for the biological activity of a-helical cytokine family proteins such as IL-6 (15, 16). These regions of GH as well as IL-6 were predicted by a mutagenesis technique to be involved in the receptor binding (17). This was confirmed later by x-ray crystallography of the GH-receptor(s) complex (18). The boundary between the CD loop and helix D in a-helical cytokines is assumed to structurally serve as an N-terminal "cap" to helix D and is called the "D1 cap region" (11). In this region, the "Dl motif' consisting of qb-(Phe or Trp)-(Glu or Gln)-(Lys or Arg)2-0-Xaa-Gly (qb = hydrophobic residue and Xaa = any residue) is proposed as the consensus sequence. The Dl motif is commonly seen, with some exceptions, in the a-helical cytokine family (Table 1). This boundary region between the CD loop and helix D is also predicted to be important for receptor binding in the case of IL-6 (21) and LIF (22). A line of evidence described above suggested that the CNTF residues at the AB loop, helix D, and the Dl cap region might be involved in receptor recognition and/or structural stability. Therefore, we analyzed the structure-function relationship of hCNTF by site-directed mutagenesis targeted on the amino acid residues in the region of the AB loop, helix D, and CD loop (especially the Dl cap region). MATERIALS AND METHODS Construction of Escherichia coli Expression Plasmid of hCNTF (pKKCNTF). The hCNTF gene was cloned from the human placenta DNA (Clontech) by the PCR method as described by Masiakowski et al. (23) with a modification. The PCR fragment encoding whole hCNTF was inserted into the BamHI and Pst I site of pKK223-4 (Pharmacia). Site-Specific Mutagenesis of hCNTF. A two-part PCR was used to create the mutation on pKKCNTF using three primers: p#N, 5'-CGGAGATCTTTTTTTATAAAATCAGGAGG-3' (at the 5' end of the hCNTF gene; plus strand); p#C(RV), 5'-CTTGCATGCATGCATGTCAGAGAAGGGAC-3' (at the 3' end of the hCNTF gene; minus strand), and the primer corresponding to the mutating site. For example, pK155 (5'C1TTTGAGAANNNCTGTGGG-3'; plus strand) was used for the Lys-155 site, or pE153 (5'-GGTCTCTTTNNNAAGAAGCTGTG-3'; plus strand) was used for the Glu-153 site; in these primers NNN indicates the codon corresponding to a substituting amino acid. These PCR products were subcloned into Abbreviations: CNTF, ciliary neurotrophic factor; CNTFR, CNTF receptor; hCNTF, human CNTF; IL, interleukin; IL-6 and IL-11, interleukins 6 and 11; DRG, dorsal root ganglion; LIF, leukemia inhibitory factor; LIFR, LIF receptor; GH, growth hormone; G-CSF, granulocyte colony-stimulating factor. *To whom reprint requests should be addressed. 8579 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Proc. Natl. Acad. Sci. USA 92 (1995) Table 1. Amino acid sequences in Dl cap region of 4-helix-bundle proteins