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. 2006 Jun 27;103(26):10068-73.
doi: 10.1073/pnas.0509598103. Epub 2006 Jun 16.

Inactivation of cardiotrophin-like cytokine, a second ligand for ciliary neurotrophic factor receptor, leads to cold-induced sweating syndrome in a patient

François Rousseau  1 , Jean-François Gauchat, James G McLeod, Sylvie Chevalier, Catherine Guillet, Florence Guilhot, Isabelle Cognet, Josy Froger, Angelika F Hahn, Per M Knappskog, Hugues Gascan, Helge Boman
Affiliations

Inactivation of cardiotrophin-like cytokine, a second ligand for ciliary neurotrophic factor receptor, leads to cold-induced sweating syndrome in a patient

François Rousseau et al. Proc Natl Acad Sci U S A. .

Abstract

Ciliary neurotrophic factor (CNTF) receptor controls a pathway supporting the differentiation and survival of a wide range of neural cell types during development and in adulthood. Cardiotrophin-like cytokine (CLC)-cytokine-like factor 1 (CLF) composite cytokine is a second ligand for the CNTF alpha-component receptor (CNTFRalpha). This composite cytokine is built on the structural model of IL-12, with a complex formed by a four-helix bundle type I cytokine, CLC (also referred to as CLCF1), bound to a soluble receptor subunit, CLF (also known as CRLF1). We have reported mutations in the chaperone soluble receptor CLF, causing cold-induced sweating syndrome (CISS). In this study, we studied the CLC-mutated alleles in a patient suffering from a similar disease. This patient was compound heterozygous for two different CLC mutations. The first allele was inactivated by a stop codon at position 107 (Y107X). In the second allele, a R197L mutation in the CLC-predicted binding site to the CNTFRalpha was detected. Functional analysis of the mutated protein revealed an incapacity for R197L CLC to bind to CNTFRalpha and activate the subsequent signaling events. Structural and docking interaction studies showed that the R197L substitution destabilized the contact site between CLC and CNTFRalpha.

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Conflict of interest statement

Conflict of interest statement: No conflicts declared.

Figures

Fig. 1.
Fig. 1.
Induced sweating test and patient description. (A) Induced sweating test. When the sublingual temperature was raised from 36.3°C to 36.9°C, mild sweating occurred only on his knees and thighs. (Left) The patient was distressed and tachypnoeic. (Right) On lowering of the sublingual temperature to 35.5°C, profuse sweating occurred on the upper trunk and arms accompanied by piloerection. (B) Patient at 46 years of age. Note the cubitus valgus and contractures of elbows, flexion contractures of fingers, and thoracolumbar scoliosis.
Fig. 2.
Fig. 2.
Identification of mutations in the CLC gene. (A) Chromatogram representing the stop mutation identified in codon 107. (B) Chromatogram representing the mutation detected in codon 197. (C) Multiple sequence alignment of CLC with CNTF and NP. Identical or similar residues are highlighted in red. Residues that are implicated in the cytokine site 1 and site 3 are boxed in black and gray, respectively. The locations of the αA-helix (blue), αA′-helix (gray), αB-helix (green), αC-helix (orange), and αD-helix (red) are indicated. Blue squares show the positions of the mutated residues in the patient.
Fig. 3.
Fig. 3.
Transcription of both CLC alleles in the patient blood cells. (A) CLC expression was analyzed by RT-PCR in peripheral blood cells. GAPDH transcripts were used as controls. (B) cDNA of patient CLC was sequenced. (Left) Coexpression of both alleles encoding either a stop and a WT codon at cDNA position 321. (Right) Coexpression of both WT and 590 G>T alleles.
Fig. 4.
Fig. 4.
Secretion and interaction of WT and mutant CLC proteins with soluble CNTFRα and CLF. Cos-7 cells were transfected with cDNAs encoding WT or mutated forms of proteins. Immunoprecipitations were performed on cell lysates or culture supernatants by using anti-CNTFRα or anti-CLF mAbs, or an anti-protein C epitope fused to CLC or ΔCLC, and proteins were analyzed by Western blotting (WB). WT and mutant CLC were detected by using a biotinylated anti-protein C tag Ab. (A) Absence of detectable expression of the truncated form of CLC (ΔCLC). WT and ΔCLC were expressed with CLF, and proteins were detected by immunoprecipitation and Western blot analysis. No protein band could be observed at the position corresponding to the expected ΔCLC molecular weight (indicated by arrows). (B) Whereas both WT and R197L CLC form secreted complexes with CLF, only WT CLC is released when coexpressed with soluble CNTFRα. The complexes were immunoprecipitated (IP) from cell culture supernatants or cell lysates, as indicated. Slight variations in CLC molecular weight represent different glycosylation states of the protein. (C) Interaction of R197L CLC and WT CLC with CLF and soluble CNTFRα were compared. Transfected cell culture supernatants were immunoprecipitated with the indicated Abs before CLC detection using an anti-protein C tag Ab.
Fig. 5.
Fig. 5.
Biological activities of WT and R197L CLC. (A) Proliferation analysis of BA/F3 GLC cell line in response to serial dilutions of purified WT (■) or R197L (●) CLC. CNTF was used as a positive control (□) (most of the standard error deviation triplicate values were inferior to the symbol size). C.P.M., counts per minute. (B) Flow cytometry analysis of the binding of WT (light gray histogram) or R197L (black histogram) CLC–CLF complexes to BAF GLC cells. Bound cytokine was revealed by using either an anti-protein C tag or an anti-CLF mAb. (C) Analysis of STAT3 Tyr phosphorylation induced by WT or the R197L CLC in the SK-N-GP human neuroblastoma cell line.
Fig. 6.
Fig. 6.
Molecular modeling and docking of CLC and CNTFRα. Aromatic residues are shown in green, positive residues are shown in blue, negative residues are shown in red, hydrophobic residues are shown in orange, and polar residues are shown in black. (A) CLC and CNTFRα are represented by blue and orange ribbons, respectively, by using pymol. (Inset) Magnified view of the boxed area of CLC-interacting site 1. (B) Motif conservation of residues implicated in site 1 of known orthologs of CLC, CNTF, and NP shown with logo.
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