Antibodies and Other Reagents
Antibodies against Cab45b were produced in New Zealand White rabbits, which were immunized with glutathione S
-transferase (GST)-Cab45b (see below) by using a standard protocol. The antibodies were affinity purified on a CNBr-Sepharose 4B column (GE Healthcare, Little Chalfont, Buckinghamshire, United Kingdom) with coupled GST-Cab45b as described previously (Laitinen et al., 2002
). The anti-SNAP23 antiserum was generated in a similar manner. The anti-Munc18b antiserum was generated as described previously (Riento et al., 1996
). The rabbit anti-syn2 and -syn3 as well as mouse monoclonal anti-tubulin were from Sigma-Aldrich (St. Louis, MO), rabbit anti-VAMP2 was from Stressgen (Nventa Biopharmaceuticals, San Diego, CA), and mouse monoclonal anti-Na+
-ATPase was from Upstate Biotechnology-Chemicon International (Temecula, CA). Rabbit antibody against Munc 18c was a gift from Dr. Y. Tamori (Kobe University, Kobe, Japan). Recombinant streptolysin-O (rSLO) was purchased from S. Bhakdi (University of Mainz, Mainz, Germany).
Yeast Two-Hybrid Screening
The full-length Madin-Darby canine kidney (MDCK)II cell Munc18b cDNA (accession no. L41609) was cloned in the GAL4 DNA binding domain bait vector pGBT9 (Clontech, Mountain View, CA), and transformed into the Saccharomyces cerevisiae strain HF7c. Interacting clones were selected from a human lymphocyte cDNA library in the pACT GAL4 activation domain vector (catalog no. HL4006AE; Clontech) by using Leu-Trp-His triple selection according to the manufacturer's instructions. Of the clones surviving the selection, those positive in an 5-bromo-4-chloro-3-indolyl-β-d-galactoside test were included for further analysis. After removal of the bait plasmid in the absence of Trp selection, the prey plasmids were isolated and transformed into Escherichia coli DH5α to produce DNA for sequencing.
Identification of Cab45 Splice Variants in the Pancreas
Initially, the National Center for Biotechnology Information sequence database was searched with the human Cab45 sequence (accession no. NM_016176), revealing a number of putative splice variants lacking exon 2, which encodes the cleavable amino-terminal signal sequence of Cab45. Thereafter, oligonucleotide primers ATATGAATTCGAAAGATGGCAGTGGCCTGATC (forward) and ATATGAATTCGCGTCGGCA ACCTCCTTCTC (reverse) annealing with human Cab45 exons 1 and 4, respectively, were designed. These primers were used to amplify and clone sequences from human pancreatic cDNA. The clones in pBluescript SK(−) (Stratagene, LaJolla, CA) were sequenced with a cycle-sequencing kit (BigDye; Applied Biosystems, Foster City, CA) and an automated ABI3730 sequencer (Applied Biosystems). This revealed cDNAs that are spliced directly from exon 1 to exon 4. To further verify the existence of such variants (denoted as b-variants) in the pancreas, a 5′ primer, GGCAGACCGGACGAGTATAAG, with nine bases from exon 1 and 12 bases from exon 4, and a 3′ primer, GGTGGGGTCCGGGACAGCC, from exon 7 (downstream of the stop codon) were used to selectively amplify b-variants from cDNAs transcribed from human total mRNAs from colon, heart, kidney, liver, lung, pancreas, and skeletal muscle (Stratagene). The reverse transcription was carried out using the above-mentioned primer that anneals with Cab45 exon 7 and the Pfu Turbo polymerase (Stratagene). To produce a cDNA for the Cab45b splice variant, the cDNA fragment encoding amino acid region M262-F362 of Cab45 was isolated by polymerase chain reaction (PCR) by using the full-length Cab45a cDNA as template and the primers ATATGGATCCATGCTCAGGTTCATGGTGAAGG and TCCGGAATTCTCAAAACTCCTCGTGCACGC. From here on, the amino acid (aa) residues of Cab45b are numbered M1-F130.
Production of Wild-Type (wt) and Mutant Cab45b Proteins in E. coli
For protein production in E. coli, the Cab45b cDNA was subcloned into the BamHI/EcoRI sites of pGEX1λT (GE Healthcare). For production of site-specifically mutated protein variants, mutagenesis was carried out on Cab45b-pGEX1λT by using the Quikchange kit (Stratagene). Point mutations were generated in EF-hand 1 (E25Q), EF-hand 2 (E70Q), and EF-hand 3 (E106Q). In addition, all three combinations of two EF-hand mutations were created. N- or C-terminally truncated variants making up aa residues D41-F130 and M1-G116, respectively, were generated by PCR, and the cDNA fragments obtained were inserted in the same vector. All constructs were verified by sequencing using a cycle sequencing kit (BigDye) and an automated ABI3730 sequencer (Applied Biosystems). The GST fusion proteins were produced in E. coli strain BL21(DE3) and purified on glutathione-Sepharose 4B (GE Healthcare) according to the manufacturer's instructions. Protein concentrations were determined by using the DC assay (Bio-Rad, Hercules, CA).
Production of His6-tagged Munc18b in Insect Cells
A recombinant baculovirus expressing His6
-Munc18b was generated and used for protein production in Sf9 cells as described previously (Riento et al., 2000
). The protein was purified on nickel-nitrilotriacetic acid agarose (QIAGEN, Valencia, CA) according to the manufacturer's instructions.
For visualization of Cab45b, rat pancreas snap frozen in liquid N2 was thawed and homogenized directly in Laemmli sample buffer containing the complete protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany), followed by incubation in a boiling water bath for 5 min. Approximately 20 μg of total protein (estimated from Coomassie-stained gels) was applied in 15% SDS-polyacrylamide gel electrophoresis (PAGE) gels, and the separated proteins were transferred onto Hybond-C Extra nitrocellulose filter (GE Healthcare). The filters were incubated with anti-Cab45b antiserum, and the bound antibodies visualized using goat anti-rabbit IgG (H+L) horseradish peroxidase conjugate (Bio-Rad) and enhanced chemiluminescence (ECL; GE Healthcare) followed by visualization by exposure to Kodak X-OMAT AR films (Eastman Kodak, Rochester, NY). For inhibition of specific Cab45b immunoreactivity, 200 μg/ml purified GST-Cab45b (see above) was incubated for 3 h at room temperature with the primary antibody, before the antibody was applied on the filter.
Lysates of rat acini containing equal amounts of protein were clarified by centrifugation (12,000 × g; 10 min). Aliquots of the resulting supernatants containing 600 μg of protein, precleared for 40 min by using 40 μl of 50% suspension of protein G-Sepharose (GE Healthcare), were incubated with Munc18b or Cab45b antibodies. Immunocomplexes were captured using 40 μl of protein G-Sepharose, which was washed four times with lysis buffer containing 1 mM Na3VO4. Samples of immunoprecipitated proteins or total cell lysates were dissolved in Laemmli buffer and boiled for 5 min. Equal amounts of protein were separated on 8% SDS-PAGE and transferred to nitrocellulose membranes (Bio-Rad), which were blocked for 1 h in Tris-buffered saline containing 5% bovine serum albumin (BSA) and then incubated with the appropriate primary antibodies. The bound antibodies were visualized using relevant peroxidase-coupled secondary antibodies and ECL.
Calcium Binding Assay
Plain GST and the purified GST-Cab45b (wild-type and all mutant/truncated proteins), 0.5 μg each, were resolved on 12.5% SDS-PAGE, electrotransferred onto Hybond-C extra nitrocellulose filter (GE Healthcare), and allowed to renature for 1 h in 60 mM KCl, 10 mM imidazole, 5 mM MgCl2, pH 6.8. The filters were probed for 10 min at room temperature with 45Ca2+ (11.63 mCi/mg; PerkinElmer, Wellesley, MA), at 1 μCi/ml in the above-mentioned buffer, washed with H2O, and exposed on Kodak X-OMAT AR films.
In Vitro Assay for Munc18–Cab45b Interaction
MaxiSorb 96-well plates (NUNC A/S, Roskilde, Denmark) were coated with GST-Cab45b (3 μg/well) in 50 mM NaHCO3
buffer, pH 9.6, for 16 h at 4°C. The binding of [35
S]methionine-labeled in vitro transcribed/translated (Tnt-coupled rabbit reticulocyte system; Promega, Madison, WI) Munc18a, Munc18b, Munc18b to the immobilized GST-Cab45b was assayed essentially as described in Riento et al. (2000)
, with the exceptions that unspecific binding was now blocked with 1% BSA, 0.05% Tween 20 in 10 mM HEPES, pH 7.2, and incubation of the in vitro-translated radioactive Munc18 proteins was carried out overnight at 4°C. Ten micromolar CaCl2
or 100 μM EGTA was added to the in vitro-translated Munc18b and to the washing buffer. For the Munc18b binding curve, 2500–250,000 cpm of the in vitro translation mixture was used. When the interactions of Munc18b, Munc18a, and Munc18c proteins were compared, equal amounts of radioactivity (100,000 cpm) were used. The numbers of methionine residues in the three proteins are rat Munc18a, 19; canine Munc18b, 15; and mouse Munc18c, 16. Background binding to wells coated with plain GST was measured in all experiments. For competition of Munc18b binding to Cab45b, 0, 1, 3, or 10 μg of His6
-Munc18b purified from insect cells was added in the in vitro-translated Munc18b aliquots (25,000 cpm) before addition in the GST-Cab45b–coated wells.
Transfection and Immunofluorescence Microscopy
The Cab45b cDNA subcloned into the mammalian expression vector pcDNA4HisMaxC (Invitrogen, Carlsbad, CA) was transfected into the Chinese hamster ovary (CHO)-K1 cell line alone or together with MDCKII Munc18b/pcDNA3.1 (Invitrogen) and/or rat syn3/pBK-CMV (Stratagene) expression plasmids, using Lipofectamine 2000 reagent (Invitrogen). After 24 h, the cells were fixed with 4% paraformaldehyde, 250 mM HEPES, pH 7.4, permeabilized with 0.05% Triton X-100/phosphate-buffered saline, and processed for indirect immunofluorescence microscopy as described previously (Johansson et al., 2005
). The bound primary antibodies were visualized using anti-mouse and anti-rabbit immunoglobulin G (IgG)-Alexa488 and -568 conjugates (Invitrogen), and the specimens were observed/images recorded with a TCS SP1 laser scanning confocal microscope (Leica, Heidelberg, Germany). Shift of the expressed Cab45b to the plasma membrane was quantified blind by visual judgment from three independent coverslips. From each double- or triple-transfected coverslip, 100 or 50 transfected cells, respectively, were analyzed.
Assay for Amylase Release from SLO-permeabilized Acini
Rat acini were prepared by collagenase digestion as described previously (Gaisano et al., 2001
). Isolated acini were suspended at 4°C in a permeabilization buffer consisting of 20 mM piperazine-N,N′
-bis-2-ethanesulfonic acid, pH 6.6, 139 mM K+
-glutamate, 4 mM EGTA, 1.78 mM MgCl2
, 2 mM MgATP, 0.1 mg/ml soybean trypsin inhibitor, 1 mg/ml bovine serum albumin, and 1 μg/ml rSLO and divided into 200-μl aliquots. rSLO does not require reducing because the single thiol group of wild-type SLO has been removed (Pinkney et al., 1989
). We have evaluated that this concentration of rSLO under the present incubation conditions induces 90–100% permeabilization as determined using trypan blue staining. Acini in rSLO-containing buffer were incubated in 4°C for a minimum of 10 min to allow toxin to partition into the plasma membrane. Excess rSLO in the supernatant was removed by low-speed centrifugation, and the pellet was resuspended in fresh permeabilization buffer at 200 μl/cell aliquot. The acini were then incubated at 37°C for 3 min to initiate pore formation. For experiments on antibody inhibition of amylase release, nonimmune rabbit IgG, total IgG from a rabbit immunized with Cab45b, or affinity-purified anti-Cab45b antibodies were added to rSLO-permeabilized acini at a final concentration of 0, 0.1, 1.0, 10, 40, or 100 μg/ml and incubated at 37°C for 30–45 min. Ca2+
-evoked amylase secretion was then induced with an equal volume of ice-cold permeabilization buffer supplemented with CaCl2
at either low (0.05 mM CaCl2
= 10 nM free Ca2+
) or high (9.8 mM CaCl2
= 10 μM free Ca2+
) concentration to reach the desired free Ca2+
level that was calculated as described previously (Kitagawa et al., 1990
). Amylase released into the supernatant during the incubation was quantified using a colorimetric method described previously and expressed as a percentage of total cellular amylase (Gaisano et al., 2001
). Total amylase is the sum of the amylase in the supernatant and acinar cell pellet, from which the stimulated amylase is expressed as a percentage of total amylase. To obtain the net stimulated release, mean basal release (10 nM Ca2+
) performed in every experiment was subtracted from mean stimulatory release (10 μM Ca2+