Worm Breeder's Gazette 14(1): 80 (October 1, 1995)
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
|1||#WUMP, The Anderson College, Glasgow University, 56 Dumbarton Road Glasgow, Scotland. email@example.com|
|2||CSHL, P.O. Box 100, Cold Spring Harbor, NY, USA.|
The cyclophilins (cyps) are a large family of ubiquitous proteins, and many different isoforms have been described in a single species (5 in man). As their name implies, these proteins bind to the immunosuppressive agent cyclosporin A (CsA). The central CsA- binding domain is commonly highly conserved, whereas the N- and C-terminal domains are relatively divergent. Many isoforms posses secretion or mitochondrial targeting sequences. The CsA/Cyp complex binds to and inhibits the protein phosphatase calcineurin, resulting in the potent T-helper cell specific immunosuppression observed in man. Biochemical studies have shown that cyps possess peptidyl prolyl cis-trans isomerase (PPIase) activity, and act both as catalysts and chaperones of protein folding, speeding up slow rate-limiting steps in the folding of various proteins, including the proline-rich collagens (Bachinger 1987; J. Biol. Chem 262, 17144). Recently, a divergent cyclophilin isoform was identified in Drosophila (ninaA) and was shown to be responsible for the specific folding of the rhodopsins in the compound eye (Stamnes et al 1991; Cell 65, 219). We are cloning and characterising the cyp isoforms from C. elegans, with the goal to determine their biological roles in nematodes. Cloning has been achieved using a combination of techniques: 1. probing cDNA libraries with degenerate genomic PCR products (corresponding to the highly conserved regions of cyclophilin); 2. obtaining full-length cDNA clones (where applicable with the aid of SL-1 RT-PCR) from the available EST- derived cyclophilin homologs; 3. isolating full-length cDNA clones corresponding to the various cyclophilin isoforms identified by the genome sequencing consortium. We have so far, identified and cloned 11 cyclophilin genes from this nematode, representing the largest number of homologs from a single species, and undoubtedly reflecting the progress of the genome sequencing project. These genes have been termed cyp-1 to -11, and where necessary their physical locations have been mapped to the YAC polytene filter (Figure 1). Several of these isoforms posses signal peptides and hence may be secreted (CYP-1, CYP-5 & CYP-6). Based on comparison to the central conserved region of human cyclophilin A, many of the worm isoforms have divergent "CsA- binding domains" (Figure 1, residues denoted * CYP-4, -8, -9 -10 & -11), an attribute hypothesised to correlate with substrate specificity (Kieffer et al 1992; J. Biol Chem 267, 5503). We have overexpressed these various isoforms in E. coli and are in the process of characterising them biochemically to determine if they posses the characteristic PPIase activity, and if so, whether this activity is sensitive to CsA. We also plan to determine the expression patterns of the various isoforms, in the hope of identifying their respective substrates, and determine their biological activity by specifically knocking out these genes via reverse genetic techniques.