Structural and Biological Characterisation of the Gut-associated Cyclophilin B Isoforms from Caenorhabditis elegans

The free-living nematode Caenorhabditis elegans expresses 18 cyclophilin isoforms, eight of which are conserved single domain forms, comprising two closely related secreted or type B forms (CYP-5 and CYP-6). Recombinant CYP-5 has been purified, crystallised and the X-ray structure solved to a resolution of 1.75 Å. The detailed molecular architecture most strongly resembles the structure of human cyclophilin B with conserved changes in loop structure and N and C-terminal extensions. Interestingly, the active site pocket is occupied by a molecule of dithiothreitol though this has little effect on the geometry of the active site which is similar to other cyclophilin structures. The peptidyl-prolyl isomerase activity of CYP-5 has been characterised against the substrate N-succinyl-Ala-Ala-Pro-Phe- p-nitroanilide, and gives a k cat/ K m value of 3.6×10 6 M −1 s −1 that compares with a value of 6.3×10 6 M −1 s −1 for human cyclophilin B. The immunosuppressive drug cyclosporin A binds and inhibits CYP-5 with an IC 50 value of 50 nM, which is comparable to the value of 84 nM found for human cyclophilin B. CYP-6 has 67% sequence identity with CYP-5 and a molecular model was built based on the CYP-5 crystal structure. The model shows that CYP-5 and CYP-6 are likely to have very similar structures, but with a markedly increased number of negative charges distributed around the surface of CYP-6. The spatial expression patterns of the cyclophilin B isoforms were examined using transgenic animals carrying a LacZ reporter fusion to these genes, and both cyp- 5 and cyp- 6 are found to be expressed in an overlapping fashion in the nematode gut. The temporal expression pattern of cyp- 5 was further determined and revealed a constitutive expression pattern, with highest abundance levels being found in the embryo.