Cell Lines, Tissues, and Antibodies
Human cervical carcinoma HeLa cells were maintained in minimal Eagle's medium (Invitrogen, Carlsbad, CA) supplemented with 8% fetal calf serum (Sigma, St. Louis, MO), 2 mM glutamax, 100 U/ml penicillin, and 100 μg/ml streptomycin (Invitrogen).
Tissues for immunoprecipitation/Western blot analysis and immunohistochemistry were obtained from rats (SD) and mice (C57/B6 or CD1). Sections of testes from human, stump-tailed macaque, and common marmoset were from blocks of fixed tissue held in an archive at the Human Reproductive Sciences Unit Edinburgh.
The anti-HA (HA-7; Sigma), anti-myc (9B11; Cell Signaling, Beverly, MA), anti-ERp72 (Calbiochem, La Jolla, CA) and anti-BiP (H-129; Santa Cruz Biotechnology, Santa Cruz, CA) antibodies were commercially available. The PDILT antiserum was raised against purified recombinant His-tagged PDILT; this antiserum was used in all experiments, except where noted. The anti-peptide PDILT antiserum was raised against the PDILT peptide RQKLINDSTNKQELN coupled to hemocyanin and was a kind gift from Dr. L. Ellgaard (University of Copenhagen, Denmark). The polyclonal antiserum against PDI has been described (Benham et al., 2000
), and the mAb against PDI was obtained from Affinity BioReagents (Golden, CO). The anti-ERp57 antiserum was a kind gift from Prof. N. Bulleid (University of Manchester, England). The calmegin antiserum was a kind gift from Prof. Y. Nishimune (The Research Institute of Microbial Diseases, Osaka, Japan). The calnexin antiserum was a kind gift from Prof. I. Braakman (University of Utrecht, The Netherlands).
Construction of human PDILT with a C-terminal myc-tag has been described previously (van Lith et al., 2005
). The PDILT cysteine-to-alanine mutants were generated from this construct using Quik Change Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA) using primers GTCAGAGCCCATCAGCGCCAAAGGAGTGGTTGAATC/GATTCAACCACTCCTTTGGCGCTGATGGGCTCTGAC (PDILT C135A) and GCACCCTGGTCTAAAAAGGCCAAGATGCTGTTCCCAC/GTGGGAACAGCATCTTGGCCTTTTTAGACCAGGGTGC (PDILT C420A). For biophysical analysis, mature PDILT (S21-L584) was cloned into pLWRP51, a modified pET23b vector (Novagen, Madison, WI), which encodes an N-terminal His tag (MHHHHHHM) before the first amino acid of the protein sequence, using the primers TTTTTTTTCATATGTCACCAGAGGTTAACGCCGGTG and TTTTTTTTGGATCCTTAT TAAAGTTCTTCCTTGACTTTTGGTTTCTTCTTTTGC. The PDILT-HA construct was a kind gift from Dr. L. Ellgaard. All plasmids generated were verified by sequencing.
Transfections with Lipofectamine 2000 (Invitrogen) were done according to the manufacturer's instructions. Subconfluent cells in 6-cm dishes were washed with Hanks' balanced salt solution and Optimem and transfected with 1 μg of DNA for 6 h in the presence of Optimem serum-free medium. After 6 h the cells were washed and returned to normal growth medium. The cells were analyzed 24 h after transfection.
RNA Isolation and RT-PCR
Total RNA from rat tissues was extracted using TRI reagent (Sigma) according to the manufacturer's instructions. The RNA concentration was determined by spectrophotometry to ensure equal total RNA input. RT-PCR was performed with the AccessQuick kit (Promega, Madison, WI) using 50 ng RNA on a Peltier Thermal Cycler (MJ Research, Waltham, MA). Sense and antisense primers were designed on different exons to exclude amplification of contaminating genomic DNA: PDILT (set 1), CATCGTTGGCTTCTTCCAG and TATTTTGGAATCTCTTCACTGG; PDILT (set 2), CCATGTGCTCAAGCAGGAAC and CACTCAGAAACTGATGTCAGGAC; calmegin, CACCTCAACCTATAGGAGAAG and CTTCATCTCATCCTCTGATCC; PDI, AAGGAATATACAGCTGGCAG and CCTTCTTCAGGCCAAAGAAC; and actin, CCACACCTTCTACAATGAGC and ACTCCTGCTTGCTGATCCAC.
PCR products were analyzed on 1% agarose gels.
Immunoprecipitations and Western Blotting
Cells and tissues were lysed in 1% Triton X-100, 50 mM Tris (pH 8.0), 150 mM NaCl, and 5 mM EDTA, supplemented with 20 mM iodo-acetic acid (IAA) and protease inhibitors. Postnuclear lysates were incubated with protein A Sepharose beads (Sigma) and antisera for 1–2 h at 4°C. After extensive washing of the beads, immunoprecipitated proteins were eluted by boiling in sample buffer and analyzed by 8% SDS-PAGE. Proteins were transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA) at 150 mA for 2 h. The membranes were blocked in Tris-buffered saline Tween (TBST) with 8% milk, followed by incubation with primary antibody at the following concentrations: 1:2000 anti-myc and anti-HA, 1:10,000 anti-PDILT, and 1:1000 anti-PDI and anti-calmegin. After washing three times with TBST, the membranes were incubated with secondary antibodies (DAKO, High Wycombe, Bucks., United Kingdom), washed, and visualized by electrochemiluminescence (ECL; Amersham/GE Healthcare, Little Chalfont, United Kingdom) and exposure to film (Eastman Kodak, Rochester, NY). Protein markers were from Bio-Rad.
Endoglycosidase H Treatment
Samples were treated with endoglycosidase H (endoH) according to the manufacturer's protocol (New England Biolabs, Beverly, MA). Briefly, lysates cleared by centrifugation were β2-mercaptoethanol–reduced and SDS-denatured followed by incubation without or with endoH in 50 mM sodium citrate (pH 5.5) at 37°C for 16 h and analyzed by SDS-PAGE.
Testicular tissues were fixed in Bouins solution, processed into paraffin wax according to standard procedures and cut in 4-μm-thick sections onto poly-lysine slides (VWR Scientific Products, West Chester, PA). Wax was removed with Histoclear (Agar Scientific, Stansted, United Kingdom), and the tissue was rehydrated in sequential washing steps from 100 to 70% ethanol. Endogenous peroxidase activity was blocked with 1% hydrogen peroxide in methanol. Antigen retrieval was done by incubating the slides in 10 mM sodium citrate at 90°C for 30 min. After a blocking step in phosphate-buffered saline (PBS) with 5% goat serum and 0.2% bovine serum albumin (BSA), the sections were incubated with primary antibodies in PBS with 0.2% BSA. Primary antibodies were detected with the ABC kit (DAKO) followed by developing with 3,3′-diaminobenzidine (DAB; Sigma). Sections were counterstained with hematoxylin. Slides were mounted with 1,3-diethyl-8-phenylxanthine (DPX; Agar Scientific) and analyzed with an inverted microscope (Axiovert 10, Zeiss, Thornwood, NY).
Purification of Human Recombinant PDILT
His-tagged PDILT was expressed in the Escherichia coli strain BL21 (DE3) pLysS in LB medium at 37°C by inducing with 1 mM isopropyl β-d-thiogalactoside at an OD600 of 0.3 for 4 h. After pelleting at 8000 rpm for 10 min, the pellet was resuspended in one-tenth volume of 20 mM sodium phosphate (pH 7.3) with 10 μg/ml DNase (Roche, Indianapolis, IN). The cells were lysed by freeze-thawing twice, and the insoluble material was collected by centrifugation (9000 rpm for 20 min). The pellet was washed twice with 20 ml of 50 mM Tris, 10 mM EDTA, 0.5% Triton X-100 (pH 8.0), twice with distilled water, and centrifuged at 9000 rpm for 20 min between each wash. PDILT was recovered from the washed pellet by solubilizing in 20 ml of 5 M guanidine hydrochloride/50 mM Tris (pH 8.75), with the insoluble material removed by centrifugation at 9000 rpm for 20 min. The soluble fraction was incubated in 10 mM dithiothreitol (DTT) at room temperature for 30 min. Excess DTT was removed by gel filtration using a PD-10 column (GE Healthcare) which had been pre-equilibrated in 5 M guanidine hydrochloride, 0.2 M sodium phosphate (pH 7.0). Solubilized His-PDILT was loaded onto a His-Trap column (GE Healthcare) and refolded on-column by a linear buffer exchange from 3 M guanidine/0.2 M sodium phosphate (pH 7.0) to 0.2 M sodium phosphate (pH 7.0) over 4 h. PDILT was eluted from the column with 50 mM EDTA, 20 mM sodium phosphate (pH 7.0).
Purified Rat PDI was a gift from N. Bulleid.
Far UV circular dichroism spectra were obtained on a Jasco J810 Spectropolarimeter (Easton, MD). Data were collected using 0.1 mg/ml PDILT at 25°C as an average of eight scans, using a cell with a path length of 0.1 cm, measured at a scan speed of 20 nm/min, a spectral bandwidth of 1.0 nm, and a time constant of 0.5 s. The maximum HT voltage was below 600 V.
Fluorescence spectra were collected on a PerkinElmer Life Sciences LS50 spectrophotometer (Boston, MA) using a 1-ml cuvette. Data were collected at 25°C as an average of four scans, excitation at 280 nm, emission at 300–400 nm, slit widths of 5 nm, and a scan speed of 200 nm/min. Protein stocks were diluted ~20-fold to a final concentration of 2 μM into 0.2 M phosphate buffer, pH 7.0, containing 0–6 M guanidine hydrochloride and equilibrated for 5 min at 25°C before fluorescence spectra were recorded. All spectra were corrected for the blank spectra with no protein added. The fluorescence parameter examined to observe the effects of guanidine hydrochloride on protein structure was the ratio of the average fluorescence intensity 2 nm either side of the λmax for native protein to the average fluorescence intensity over the range 320–400 nm. This parameter was chosen because it is independent of concentration and less dependent on the direct effects of guanidine hydrochloride on tryptophan fluorescence.
For the insulin-reduction assay, human insulin (Sigma) at a final concentration of 0.17 mM was incubated with 1 μM PDILT or PDI in the presence of 5 mM DTT. Precipitation of the insulin B chain was monitored by measuring OD600 at 5-min intervals after addition of insulin.
To monitor protein oxidation and isomerization, 70 μM of recombinant bovine pancreatic trypsin inhibitor (BPTI) in 0.1 M Tris buffer, pH 7.5, or 0.1 M phosphate buffer, pH 6.0, both containing 1 mM EDTA, 2 mM reduced glutathione (GSH), and 0.5 mM oxidized glutathione was incubated with and without 10 μM of PDILT. Disulfide bond formation of BPTI was monitored by trapping thiol-disulfide exchange by treating with 1.1 M iodoacetamide, desalting the sample, and analyzing it by electrospray mass spectrometry (Micromass, Manchester, United Kingdom).
The protein oxidation assay using a fluorescent decapeptide with 3.2 μM peptide substrate and 0.2 μM PDILT has been described (Ruddock et al., 1996
). Deglutathionylation activity was monitored as described previously (Peltoniemi et al., 2006
), using 0.2 μM PDILT in the presence of 1 mM GSH and 5 μM substrate peptide. Glutathionylation activity was determined as described previously (Peltoniemi et al., 2006
), using 0.2 μM PDILT and 5.4 μM substrate peptide.
Cell extracts from E. coli
BL21 (DE3) pLysS were prepared by repeated freeze-thawing. Bolton-Hunter 125
I-labeling of Δ-somatostatin (AGSKNFFWKTFSS) was performed as recommended by the manufacturer (Amersham Pharmacia Biotech, Piscataway, NJ). Cross-linking was performed using the homobifunctional cross-linking reagent disuccinimidyl glutarate (Sigma) as described (Klappa et al., 1998a
). As a positive control an E. coli
lysate expressing ERp27 (Alanen et al., 2006
) was used. This lysate was prepared as detailed for the purification of PDILT, except that because ERp27 is solubly expressed, the soluble postlysis material was used.