Abstract
Cyclophilin 3 (CYP-3) is one of the most abundantly expressed cyclophilin isoforms in the free living nematode Caenorhabditis elegans. The detailed post-embryonic expression pattern of the cyp-3 transcript is unusual, peaking during early larval development. The spatial expression pattern was examined via reporter gene analysis demonstrating that the cyp-3 transcript is exclusively expressed in the single anterior excretory cell. Recombinant cyclophilin 3 has been purified, crystallized and solved to a resolution of 1.8 A. The peptidyl-prolyl isomerase activity of CYP-3 has been characterized against the substrate N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, and gives a k(cat)/K(m) value of 2.4 x 10(6) M(-1) s(-1). The immunosuppressive drug cyclosporin A binds and inhibits CYP-3 with an IC(50) value of 16 nM, comparable with the range of values found for human cyclophilin A. The x-ray structure shows that the overall fold and active site geometry is similar to other cyclophilin structures. There are however a number of distinctive features, and we use this structure and amino acid sequence alignment analysis to identify a subgroup of "divergent-loop cyclophilins". This subgroup has a number of uniquely conserved features: an additional loop between residues 48 and 54 (KSGKPLH); two cysteine residues (Cys(40) and Cys(168)) that are in close proximity but remain in the unoxidized form, and two other conserved residues, His(54) and Glu(83). We suggest that these features are functionally important for the role played by this class of cyclophilins during cellular responses to stress caused by changes in the redox environment or by up-regulation of cellular activity. This study represents a detailed biological, biochemical, and structural characterization of a single cyclophilin isoform in the model organism Caenorhabditis elegans.
Highlights
MethodsConstruction of C. elegans CYP-3 Reporter Gene Fusions—The cyp-3 reporter gene recombinant plasmids were constructed using the C. elegans lacZ promotorless reporter gene expression vector pPD95.03 (a kind gift from Andy Fire and co-workers, Carnegie Institute)
Sodium citrate, pH 6.0, 13% MPEG 5000
Cell pellets were frozen at Ϫ20 °C, defrosted on ice, and solubilized in lysis buffer (10% w/v) containing 50 mM HEPES, 5 mM benzamidine, 5 mM EDTA, 5 mM 2-mercaptoethanol, pH 7.5
Summary
Construction of C. elegans CYP-3 Reporter Gene Fusions—The cyp-3 reporter gene recombinant plasmids were constructed using the C. elegans lacZ promotorless reporter gene expression vector pPD95.03 (a kind gift from Andy Fire and co-workers, Carnegie Institute). The plasmid encodes a nuclear localization signal amino-terminal to the lacZ gene and an N-terminal multi-intron intervening sequence. The translationally “in-frame” 1026-base pair 5Ј untranslated region of the cyp-3 gene was amplified from genomic DNA by polymerase chain reaction (PCR) with Taq polymerase (Applied Biotechnologies). The PCR primers contained an artificial restriction site (underlined) to allow directional subcloning into the multiple cloning site of the vector pPD95.03; cyp3pfxba 5Ј-gcgtctagagaagtccaaattactgtcaac-3Ј (sense, XbaI); cyp3prbam 5Ј-gcgggatccctcattgttgaggcaagaataggga-3Ј (antisense, BamHI). This construct permitted an in-frame translational fusion to the lacZ with the first two amino acids of CYP-3. Miniprep DNA was prepared using a Qiagen miniprep kit, and sequencing was performed to confirm the identity and the translational context of the insert
Sign-in/Register to view highlights & summary 
Published Version
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call