Lemon protein disulfide isomerase: cDNA cloning and biochemical characterization
© Chen et al.; licensee Springer. 2013
Received: 27 May 2013
Accepted: 12 September 2013
Published: 16 September 2013
Protein disulfide isomerases (PDIs), a family of structurally related enzymes, aid in protein folding by catalyzing disulfide bonds formation, breakage, or isomerization in newly synthesized proteins and thus.
A ClPDI cDNA (1828 bp, GenBank accession HM641784) encoding a putative PDI from Citrus limonum was cloned by polymerase chain reaction (PCR). The DNA sequence encodes a protein of 500 amino acids with a calculated molecular mass of 60.5 kDa. The deduced amino acid sequence is conserved among the reported PDIs. A 3-D structural model of the ClPDI has been created based on the known crystal structure of Homo sapiens (PDB ID: 3F8U_A). The enzyme has two putative active sites comprising the redox-active disulfides between residues 60–63 and 405–408 (motif CGHC). To further characterize the ClPDI, the coding region was subcloned into an expression vector pET-20b (+), transformed into E. coli Rosetta (DE3)pLysS, and recombinant protein expressed. The recombinant ClPDI was purified by a nickel Sepharose column. PDI’s activity was assayed based on the ability of the enzyme to isomerize scrambled RNase A (sRNase A) to active enzyme. The KM, kcat and kcat/KM values were 8.3 × 10-3 μM, 3.0 × 10-5 min-1, and 3.6 × 10-1 min-1 mM-1. The enzyme was most active at pH 8.
The advantage of this enzyme over the PDI from all other sources is its low KM. The potential applications of this PDI in health and beauty may worth pursuing.
KeywordsCitrus limonum Protein disulfide isomerase (PDI) Scrambled RNase A (sRNase A) Three-dimension structural model (3-D structural model)
Protein disulfide isomerase (PDI), a member of thioredoxin-superfamily, plays a key role in catalyzing disulfide bond formation, reduction, and isomerization. It was the first reported protein folding enzyme (Venetianer and Straub 1963a1963b; Goldberger et al. 31963). PDI is best known as resided in endoplasmic reticulum (ER) where folding, modification, and quality control of many secretory and cell-surface proteins take place (Hatahet and Ruddock 2009; Lambert and Freedman 1983). In vivo, native disulfide bond formation involves the formation of new disulfides (oxidation) and the rearrangement of non-native disulfides (isomerization). PDI, contains combinations of catalytically active and inactive thioredoxin domains, is capable of catalyzing both the oxidation of new disulfides and the isomerization of existing disulfides (Kulp et al. 2006). The first and last domains (referred to as a and a’ domain, respectively) contain CxxC active site motifs, whereas the two middle domains (referred to as b and b’ domain) are catalytically inactive (Kemmink et al. 1997). Oxidation, catalyzed by the a’ domain, involves the transfer of an active site disulfide from PDI to substrate proteins. Isomerization, catalyzed by the a domain, requires the active site cysteines to be in the reduced form in order to attack non-native disulfides in substrate proteins thereby catalyzing their rearrangement (Kulp et al. 2006).
Like other proteins with thioredoxin folds, PDI is a multifunctional protein (Pedone et al. 2010). In addition to catalyze protein folding, PDI is the β-subunit of prolyl-4-hydroxylase (Koivu et al. 1987), is a subunit of microsomal triglyceride transfer protein (Wetterau et al. 1991), acts as a chaperone (Wang and Tsou 1993), is implicated in peptide loading onto MHC class I (Peaper and Cresswell 2008), and is involved in regulating NAD(P)H oxidase (Janiszewski et al. 2005). Although members of PDI enzyme have a classic ‘KDEL’ ER retention signal at the C-terminal end (Pelham 1990), the enzymes have been identified outside the ER at many subcellular locations and are known to involve in a wide range of biological functions (Turano et al. 2002). Outside of ER, PDI has been reported to serve as switches for modulating protein function in some cases (Hogg 2003; Wouters et al. 2010). For instance, extracellular PDI mediated disulfide exchange has been shown to switch tissue factor between coagulation to cell signaling (Wouters et al. 2010; Ahamed et al. 2006).
Lemon is an economically valuable produce in Taiwan. It is an amazing fruit that has been used as natural remedy for health and beauty. PDI has been used as an ingredient in cosmetic agents for treating hair (European Patent WO2003099243 A1). Here, we report the cloning of a putative PDI cDNA from lemon, namely ClPDI. The coding region of the ClPDI cDNA was introduced into an E. coli expression system and the active enzyme purified and characterized. Understanding the properties of this ClPDI will be beneficial for its potential applications such as catalyzing protein folding or serving a chaperone contributing to reactivation of inactive enzymes.
Total RNA preparation from lemon and cDNA synthesis
A fresh lemon (Citrus limonum) was obtained from a local market. The lemon including the skin (4 g) was frozen in liquid nitrogen and ground to powder in a ceramic mortar. PolyA mRNA (35 μg) was prepared using Straight A’s mRNA Isolation System (Novagen, USA). Four μg of the mRNA was used in the 5′-RACE-Ready cDNA and 3′-RACE-Ready cDNA synthesis using Clontech’s SMART RACE cDNA Amplification Kit.
Isolation of ClPDI cDNA
Using the 5′-RACE-Ready cDNA of lemon as a template and two degenerate primers (5′ AGY CAA GGT GCH TTC CAG 3′ and ACY TTM ACW GGC TCR TTG TT), a 0.2 kb fragment was amplified by PCR. The degenerate primers were designed based on the conserved sequences of PDI from AtPDI (Arabidopsis thaliana, AY063059), HsPDI (Homo sapiens, 3F8U_A), MmPDI (Mus musculus, 2DJ2_A), HiPDI (Humicola insolens, 2DJJ_A). The 0.2 kb fragment was subcloned and sequenced. On the basis of this DNA sequence, two primers near both ends, a ClPDI-6R primer (5′ ACY TTM ACW GGC TCR TTG TT 3′) and a ClPDI-7 F primer (5′ GCA CCT TGG GTG AAG GAA TAC 3′) were synthesized. The primers allowed sequence extension from both ends of the 0.2 kb fragment when used with the UPM primer (universal primer A mix, purchased from BD biosciences). Two PCRs were carried out each using 0.1 μg of the 5′-RACE-Ready cDNA or 3′-RACE-Ready cDNA as a template. The primer pairs in each reaction were UPM and ClPDI-6R primers, and UPM and ClPDI-7 F primers. A 1.3 kb fragment (5′-RACE; 5′-DNA end) and a 1.0 kb DNA (3′-RACE; 3′-DNA end) were amplified by PCR. Both DNA fragments were subcloned into pCR4 vector and transformed into E. coli TOPO10. The nucleotide sequences of these inserts were determined in both strands. Sequence analysis revealed that the combined sequences covered an open reading frame of a putative ClPDI cDNA (1828 bp, EMBL no. HM641784). The identity of this ClPDI clone was assigned by comparing the inferred amino acid sequence in various data banks using the basic local alignment search tool (BLAST).
Bioinformatics analysis of ClPDI
The BLASTP program was used to search homologous protein sequences in the nonredundant database (NRDB) at the National Center for Biotechnology Information, National Institutes of Health (http://www.ncbi.nlm.nih.gov/). Multiple alignments were constructed using ClustalW2 program. Protein secondary structure was predicted by SWISS-MODEL program and represented as α helices and β strands. A 3-D structural model of PDI was created by SWISS-MODEL (http://swissmodel.expasy.org/) based on the known crystal structure of Homo sapiens PDI (PDB code 3F8U_A) (Dong et al. 2009). The modeling data was then superimposed with that of PDB ID: 3F8U_A by DeepView Swiss-PdbViewer v4.1 (http://spdbv.vital-it.ch/).
Subcloning of ClPDI cDNA into an expression vector
The coding region of the ClPDI cDNA was amplified using gene specific flanking primers. The 5′ upstream primer contains Eco RI recognition site (5′ CGT CTC GAA TTC GAT GGC CAG TCG ATC GAT 3′) and the 3′ downstream primer contains Not I recognition site (5′ GCG GCC GCG AGC TCA TCT TTT CCA GA 3′). Using 0.2 μg of ClPDI cDNA as a template, and 10 pmole of each 5′ upstream and 3′ downstream primer, a 1.5 kb fragment was amplified by PCR. The fragment was ligated into pCR4 and transformed into E. coli. The recombinant plasmid was isolated and double digested with Eco RI and Not I. The digestion products were separated on a 1.0% agarose gel. The 1.5 kb fragment was gel purified and subcloned into the Eco RI and Not I site of pET-20b(+) expression vector (Novagen). The recombinant DNA was then transformed into E. coli Rosetta (DE3)pLysS and protein expressed by isopropyl β-D-thiogalactopyranoside (IPTG) induction. Expression of functional recombinant protein was demonstrated by enzyme activity assay as described below.
Expression and purification of the recombinant ClPDI
The transformed E. coli containing the ClPDI gene was grown at 37°C in 200 mL of Luria-Bertani containing 50 μg/mL ampicillin until A 600 reached 0.6. Protein expression was induced by the addition of IPTG to a final concentration of 50 μM. The culture was incubated at 80 rpm for an additional 2 h at 32°C. The cells were harvested and soluble proteins extracted in PBS with glass beads as described before (Ken et al. 2005). The recombinant ClPDI was purified by Ni-NTA affinity chromatography (elution buffer: 30% PBS containing 20–250 mM imidazole) as per the manufacture’s instruction (Qiagen). The purified protein was checked by a 10% SDS-PAGE. Protein bands on gel were visualized by staining with Coomassie Brilliant Blue R-250. Protein concentration was determined by a Bio-Rad Protein Assay Kit (Richmond, CA) using bovine serum albumin as a reference standard.
Molecular mass analysis via electrospray ionization quadrupole-time-of-flight (ESI Q-TOF)
The purified recombinant ClPDI (0.21 mg/mL) was prepared in 0.1 mM Tris–HCl containing 0.05 mM NaCl, 0.5 mM imidazole and 0.03% glycerol. The sample (5 μL) was used for molecular mass determination using an ESI Q-TOF mass spectrometer (Micromass, Manchester, England).
ClPDI activity assay
PDI activity was assayed by the method of Ibbetson and Freedman (1976) using scrambled ribonuclease (sRNase A) as a substrate. In this method, PDI was used to activate sRNase A then the ribonuclease activity was monitored spectrophotometriclly as described below. Each 5 μL ClPDI sample (0.2 μg/μL stock solution) was first pre-incubated with 10 μL of 100 μM DTT (dithiothreitol) for 5 min at 25°C. Next, a 0–60 μL portion of the sRNase A (0.036 μM) was added, followed by addition of 10 μL of 0.5 M Tris/HCl, pH 7.5 and appropriate amount of H2O to 90 μL. The mixture was incubated for 20 min at 25°C to allow conversion of sRNase A to active RNase by PDI. The RNase activity was then measured by its ability to degrade RNA. Ten μL of RNA (2 μg/μL) was added to each assay mixture (Total volume was 100 μL). The samples were incubated at 37°C for another 5 min. Three hundred μL of 95% ethanol was added to each assay mixture to precipitate the residual RNA. Ribonuclease activity was monitored by observing the decrease in A 260 of the residual RNA.
The kinetic properties of the ClPDI (1.0 μg) was determined by varying the concentrations of sRNase A (3.6 to 21.6 nM) with fixed amount of 20 μg RNA (2 μg/μL). The change in absorbance at 260 nm was recorded between 1 and 20 min. The KM, Vmax and kcat were calculated from Lineweaver-Burk plots.
The ClPDI enzyme was tested for stability in terms of its activity under various conditions. Aliquots of the CIPDI sample (1.0 μg) were treated as follows: (1) Thermal effect. Each enzyme sample (1.0 μg) was heated at 37, 50, 60, 70, or 80°C for 20 min. (2) pH effect. Each enzyme sample (1.0 μg) was adjusted to desired pH by adding a half volume of buffer with different pHs: 0.2 M citrate buffer (pH 2.5, or 4.0), 0.2 M phosphate buffer (pH 6.0, 7.0 or 8.0) or 0.2 M CAPS buffer (pH 10.0, or 11.0). Each sample was incubated at 37°C for 1 h. (3) Imidazole effect. During protein purification, the CIPDI enzyme was eluted with imidazole, therefore, its effect on activity was examined. Imidazole was added to each enzyme sample to the final levels of 0.2, 0.4, 0.8 or 1.0 M and incubated at 37°C for 1 h. (4) DTT effect. DTT was added to each enzyme sample to the final levels of 10, 30, 70, 100 or 200 μM and incubated at 37°C for 5 min. At the end of each treatment, samples were checked for CIPDI activity.
Results and discussion
Cloning and characterization of a cDNA encoding ClPDI
Expression and purification of the recombinant ClPDI
Kinetic studies of the purified ClPDI
Kinetic characterization of ClPDI and that from other sources
The kcat value of ClPDI is also several orders of magnitude smaller than that of PDI from other sources (Table 1). But its kcat/KM value is compatible to that of PDI from other sources. It is likely that the ClPDI is one of the early enzymes that responsible for oxidative folding of proteins in lemon as the enzyme can respond to extremely low substrate concentration.
Characterization of the purified ClPDI
The importance of PDI has been implicated in health and disease (Benham 2012; Andreu et al. 2012). This study reported the first cloning and expression of an important protein folding enzyme, ClPDI, from lemon. The active form of the ClPDI has been successfully expressed in E. coli and characterized. The advantage of this enzyme over the PDI from all other sources is its extremely low KM. The potential applications of this enzyme in health and beauty may worth pursuing.
Protein disulfide isomerase
- sRNase A:
Scrambled RNase A
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
Phosphate buffer saline.
This work was supported by the National Science Council of the Republic of China, Taiwan under grant NSC 100-2313-B-019-003-MY3 to C-T. Lin.
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