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.

Cloning and Characterization of the C. elegans Cyclophilin Isoforms

Antony Page¹, Kenny MacNiven¹, Micheal Hengartner², Iain Johnstone¹

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.