Molecular and Biochemical characterization of the Parvulin-type PPIases in Lotus

The cis/trans isomerization of the peptide bond preceding proline is an intrinsically slow process, although important in many biological processes in both prokaryotes and eukaryotes. In vivo this isomerization is catalyzed by peptidyl-prolyl cis/trans isomerases (PPIases). Here we present the molecular and biochemical characterization of parvulin-type PPIase family members of the model legume Lotus japonicus , annotated as Lj Par1, Lj Par2 and Lj Par3. Although Lj Par1 and Lj Par2 were found to be homologous to PIN1-type parvulins and h Par14 from human, respectively, Lj Par3 represents a novel multi-domain parvulin, apparently present only in plants, which contains an active C-terminal sulfurtransferase domain. All Lotus parvulins were heterologously expressed and purified from Escherichia coli , and purified protein verification measurements where contacted an LC-MS based proteomic method. The biochemical characterization of the recombinant Lotus parvulins revealed that they posses PPIase activity towards synthetic tetrapeptides, although they exhibited different substrate specificities depending on the amino acid N-terminal to proline. These differences were also studied in a structural context using molecular modeling of the encoded polypeptides. Real-time RT-PCR revealed that the three parvulin genes of Lotus are ubiquitously expressed in all plant organs. Lj Par1 was found to be upregulated during the later stages of nodule development. Subcellular localization of Lj Par-eYFP fusions expressed in Arabidopsis leaf epidermal cells revealed that Lj Par1- and Lj Par2-eYFP fusions were localized in the cytoplasm and in the nucleus, in contrast to Lj Par3-eYFP which was clearly localized in plastids. Divergent substrate specificities, expression profiles, and sub-cellular localization indicate that plant parvulin-type PPIases are probably involved in a wide range of biochemical and physiological processes. characterized three parvulin-type PPIases from the model legume L. japonicus . To our knowledge, this is the first report of a family-wide study of the plant parvulins. Our results revealed different substrate specificities of the three enzymes, which probably reflects differences in their natural targets in planta . The ubiquitous presence of PPIase in plants, together with the presence of the novel Lj Par3 homologues, underscores the importance of prolyl cis/trans isomerization in various biological processes during plant life. Further work needs to be done to clarify the processes which involve PPIase-dependent prolyl cis/trans isomerization in plants. To this end, we plan to use reverse genetics, including TILLING and RNA interference to ascertain the function of parvulin-type PPIase in L. japonicus . sections were cut. Antisense and sense RNA probes labelled with digoxigenin-11-rUTP (Roche) were transcribed from the respective cDNA clones using the appropriate RNA polymerase promoters on the plasmid vectors. The probes were partially degraded to an average length of 150 nucleotides. Sections were hybridized overnight at 42 o C in 50% (v/v) formamide, 300 mM NaCl, 10 mM Tris-HCl pH 7.5, 1 mM EDTA, 0.02% (w/v) Ficoll, 0.02% (w/v) polyvinylpyrrolidone, 0.025% (w/v) bovine serum albumin (BSA), 10% (v/v) dextran sulfate and 60 mM dithiothreitol (DTT). Hybridization signals were visualized with anti-digoxigenin antibodies conjugated with alkaline phosphatase. Images were processed and prepared for presentation with Photoshop 7 software (Adobe Systems Inc., San Jose, CA, USA).