Journal of Bacteriology, September 2004, p . 6325-6326, Vol . 186, No . 18

Recombinant Cyclophilins Lack Nuclease Activity

Angel Manteca and Jesus Sanchez^*

Departamento de Biología Funcional, Facultad de Medicina, Universidad de Oviedo, Oviedo, Spain

Received 7 April 2004/ Accepted 9 June 2004

 
Several single-domain prokaryotic and eukaryotic cyclophilinshave been identified as also being unspecific nucleases witha role in DNA degradation during the lytic processes that accompanybacterial cell death and eukaryotic apoptosis . Evidence is providedhere that the supposed nuclease activity of human and bacterialrecombinant cyclophilins is due to contamination of the proteinsby the host Escherichia coli endonuclease and is not an intrinsicproperty of these proteins.

 
Some reports on human recombinant CypA, CypB, and CypC cyclophilins[6], murine CypB cyclophilin [8], Legionella pneumophila LpCyp18 cyclophilin [12], and Streptomyces antibioticus-SanCyp18 cyclophilin[9] describe associated Ca²⁺/Mg²⁺ nuclease-dependent activity.For the aforementioned eukaryotic cyclophilins, the accompanyingnuclease activity has been proposed as playing a central rolein apoptosis . This is the case with NUC-18, a nuclease reportedto be identical to rat CypA cyclophilin, involved in glucocorticoid-stimulatedapoptosis of thymocytes [7] and murine CypB cyclophilin-associatednuclease, involved in TCR-stimulated apoptosis of thymocytes[8] . For S . antibioticus SanCyp18 cyclophilin, nuclease activityhas been related to chromosomal DNA degradation during the lysisof substrate [vegetative] mycelium [2], which precedes the emergence of the aerial [reproductive] mycelium [9] . Amino acid sequenceanalysis of the S . antibioticus SanCyp18 protein identifiedit as a typical cyclophilin that is clearly homologous to cyclophilinsfrom gram-negative bacteria [A . Manteca, T . Kamphausen, J . Fanghanel,G . Fischer, and J . Sanchez, submitted for publication] . Thebiochemical characteristics of SanCyp18 recombinant cyclophilin[binding and inhibition by cyclosporine A and structure of theloop region of the protein] were analogous to those of gram-negativebacteria cyclophilins, from which it may have been passed toStreptomyces species [Manteca et al., submitted].

To analyze the hypothetical associated nuclease activity, SanCyp18 cyclophilin was expressed in Escherichia coli JM109[DE3] [endA1 recA1 gyrA96 thi hsdR17 [r_K^– m_K⁺] relA1 supE44 ^– [lac-proAB] [F' traD36 proAB lacI^qZM15] DE3], a strain lackingperiplasmic endonuclease I [14] . The nuclease activity of recombinantSanCyp18 was further detected by activity in gel assays, asdescribed formerly [9] . The nucleases were separated in a 12%gel containing 10 µg of denatured calf thymus DNA [Sigma]/mlby sodium dodecyl sulfate [SDS] [USB-US75819; Amersham Biosciences]-polyacrylamidegel electrophoresis [PAGE] [9] . After electrophoresis, the proteinswere renatured by repeatedly washing the gel with renaturationbuffer [25 mM Tris-HCl [pH 8.8], 1 mM EDTA, 7 mM ß-mercaptoethanol]during 2 h at 4°C . Nuclease activity was visualized by incubatingthe gels for 1 h at 37°C in 20 mM Tris-HCl [pH 8.0]-7 mM2-mercaptoethanol-10 mM MgCl₂-5 mM CaCl₂-10% dimethyl sulfoxidebuffer, as reported elsewhere [9], followed by staining withethidium bromide and analysis under UV light . Micrococcal nucleaseand bovine pancreatic DNase I were included as positive controls.As can be seen in Fig . 1, the recombinant protein overexpressedin E . coli JM109[DE3] lacks nuclease activity.

 
The results obtained with S . antibioticus SanCyp18 cyclophilin prompted us to extend the analysis to human CypA and L . pneumophila LpCyp18 cyclophilins, which were also reported to be nucleases by SDS-PAGE activity analysis [see above] . We used several E. coli strains as hosts to test the putative nuclease activity for recombinant human CypA [7] and recombinant LpCyp18 [12]cyclophilin . E . coli NM522 and M15 strains were used in thestudies mentioned above to express human CypA and LpCyp18, respectively[7, 12] . Both of them possess the aforementioned periplasmic endonuclease I, which was absent from the E . coli JM109 strain used to express SanCyp18 . This nuclease is detected as a clear band in activity gel assays with E . coli M15 cell extracts [Fig. 1, center panel, lane C] . We expressed human CypA and LpCyp18cyclophilins genes in E . coli M15 [pREP4] [endA⁺] and E . coliJM105 [pREP4] [endA mutant] [14] by the use of pTTE1 expressionvector [12] for the LpCyp18 gene and pTTE2 expression vector[Thomas Tradler, Max-Planck Forschungsstelle "Enzymologie der Proteinfaltung," Halle/Saale, Germany] for the human CypA geneto investigate the hypothetical associated nuclease activity.The plasmids were introduced in the aforementioned strains tooverexpress these proteins, following the protocols describedfor the QIAGEN system employed . An activity gel assay and SDS-PAGEanalysis of the proteins were carried out with the crude E.coli extracts . Although the levels of overexpression were similarin both E . coli strains [Fig . 1], nuclease activity was only observed in the wild-type M15 [endA⁺] strain . Moreover, the M15 strain extract without any expression vector showed the same nuclease activity level as the M15 strain extracts withthe expressed cyclophilins . Finally, the sizes of nuclease andhuman cyclophilin are different [Fig . 1, center panel, lane H] . A previous report showed that, at least for the L . pneumophila LpCyp18 cyclophilin, the hypothetical associated nuclease activity was not linked with its native structure [12] . These resultsare coherent with our finding that the nuclease activity is caused by a different protein . The overall data show that, at least for the recombinant cyclophilins, the E . coli endonuclease is responsible for the activity seen on the gels; therefore, this cannot be attributed to an intrinsic property of cyclophilins. On the other hand, as CsA is a competitive tight-binding inhibitor, it does not inhibit cyclophilin-associated nuclease activity[6] . Cyclophilin residues involved in PPIase activity and inCsA binding have been well characterized [3, 5, 10, 11] . Boththe PPIase domain and the CsA binding sites span the completelength of the amino acid sequence and build a cleft in the coreof the three-dimensional structure . As the PPIase active siteis efficiently blocked by CsA, an additional nuclease domainwould be required to harbor nuclease activity . The small sizeof most characterized cyclophilins makes such an additionaldomain very unlikely.

E . coli endonuclease I is a very active periplasmic enzyme whose function is unknown [1, 13] . The protein has a putative signalpeptide for a cleavage site that would give rise to a matureprotein of about 24 kDa [4], consistent with the molecular massobserved in the gel [Fig. 1, center panel, lane C] . The 18-kDa Streptomyces nuclease [9] and NUC18 [7] enzymes both exhibitlow substrate specificity and high enzymatic activity that canbe visualized using SDS-PAGE activity gel assays, even at verylow amounts that are not detectable on silver-stained SDS-PAGE[A . Manteca and J . Sanchez, unpublished data] . Minor contaminationof cyclophilins with these or other nucleases can lead to erroneouslyassociating these activities with the cyclophilin proteins,as occurred in the case of SanCyp18 and LpCyp18 human recombinantcyclophilins . Other native cyclophilins described as nucleases,such as NUC18 [7], murine Cyp B [8], and SanCyp18 [9], mightalso conceivably be contaminated by host activities . The presenceof nuclease-contaminated cyclophilin samples could equally explainsome published results on the inhibition of cyclophilin-associated nuclease activity by cyclophilin antibodies [8] . In summary, it can be concluded that SanCyp18 and human and Legionella recombinantcyclophilins lack nuclease activity; consequently, other publisheddata concerning the presence of this activity in native cyclophilinsshould be analogously reappraised.

 
We are grateful to Thomas Tradler, who provided the human CypAclone, and to Paul Barnes for revising the text.

This research was supported by grant BIO2000-0577 from the DGI, Subdirección General de Proyectos de Investigación,MCYT, Madrid, Spain.

 
* Corresponding author . Mailing address: Universidad de Oviedo, Departamento de Biología Funcional, Area de Microbiologia, Julian Claveria s/n, Oviedo 33006, Spain . Phone: 34 985103555 . Fax: 34 985103148 . E-mail: jsm@fq.uniovi.es .

1. Durwald, H., H . and Hoffmann-Berling. 1968 . Endonuclease-I-deficient and ribonuclease I-deficient Escherichia coli mutants . J . Mol . Biol . 34:331-346.
2. Hodgson, D . A. 1992 . Differentiation in Actinomycetes, p . 407-440 . In S . Mohan, C . Dow, and J . A . Cole [ed.], Prokaryotic structure and function: a new perspective, vol . 47 . Society for General Microbiology Symposium . Cambridge University Press, Cambridge, United Kingdom.
3. Ivery, M . T . G. 2000 . Immunophilins: switched on protein binding domains? Med . Res . Rev . 20:452-484.
4. Jekel, M., and W . Wackernagel. 1995 . The periplasmic endonuclease I of Escherichia coli has amino-acid sequence homology to the extracellular DNases of Vibrio cholerae and Aeromonas hydrophila . Gene 154:55-59.
5. Kallen, J., C . Spitzfaden, M . M . G . Zurini, G . Wider, H . Widmer, K . Wülthrich, and D . M . Walkinshaw. 1991 . Structure of human cyclophilin and its binding site for cyclosporin A determined by X-ray crystallography and NMR spectroscopy . Nature 353:276-279.
6. Montague, J . W., F . M . Hughes., and J . A . Cidlowski. 1997 . Native recombinant cyclophilins A, B and C degrade DNA independently of peptidylprolyl cis-trans-isomerase activity . J . Biol . Chem . 272:6677-6684 .
7. Montague, J . W., M . L . Gaido, C . Frye, and J . A . Cidlowski. 1994 . A calcium-dependent nuclease from apoptotic rat thymocytes is homologous with cyclophilin . J . Biol . Chem . 269:18877-18880 .
8. Nagata, T., H . Kishi, Q . L . Liu, T . Yoshino, T . Matsuda, Z . X . Jin, K . Murayama, K . Tsukada, and A . Muraguchi. 2000 . Possible involvement of cyclophilin B and caspase-activated deoxyribonuclease in the induction of chromosomal DNA degradation in TCR-stimulated thymocytes . J . Immunol . 165:4281-4289 .
9. Nicieza, G . R., J . Huergo, B . A . Connolly, and J . Sánchez. 1999 . Purification, characterization, and role of nucleases and serine proteases in Streptomyces differentiation . J . Biol . Chem . 274:20366-20375 .
10. Plügl, G., J . Kallen, T . Schirmer, N . J . Jansonius, M . M . G . Zurini, and D . M . Walkinshawm. 1993 . X-ray structure of a decameric cyclophilin-cyclosporin crystal complex . Nature 361:91-94.
11. Schiene-Fischer, C., J . Habazettl, F . X . Schmid, and G . Fischer. 2002 . The hsp70 chaperone DnaK is a secondary amide peptide bond cis-trans isomerase . Nat . Struct . Biol . 9:419-424.
12. Schmidt, B., T . Tradler, J . U . Rahfeld, B . Ludwing, B . Jain, K . Mann, K . P . Rücknagel, B . Janowski, A . Schierhorn, G . Küllertz, J . Hacker, and G . Fischer. 1996 . A cyclophilin-like peptidyl-prolyl cis/trans isomerase from Legionella pneumophila-characterization, molecular cloning and overexpression . Mol . Microbiol . 21:1147-1160.
13. Taylor, R . G., D . C . Walker, and R . R . McInnes. 1993 . E . coli host strains significantly affect the quality of small scale plasmid DNA preparations used for sequencing . Nucleic . Acids . Res . 21:1677-1678.
14. Yanisch-Perron, C., J . Vieira, and J . Messing. 1985 . Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors . Gene 33:103-119.

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