Nylon filters containing polyadenylated RNAs from human tissues and tumor cell lines were purchased from Clontech. Restriction endonucleases and different reagents for molecular cloning, including the Expand™ High Fidelity PCR system and the Thermoscript reverse transcription-PCR system were from Roche Applied Science. DNA probes were radiolabeled using [α-32P]dCTP (3000 Ci/mmol) and a random-priming kit from Amersham Biosciences.
Bioinformatic analysis and cDNA cloning
Human polyserase-2 cDNA and the BLAST program were used to query regions in the human genome that could be predicted as new polyserase genes. These searches allowed us to identify a putative region in chromosome 16p11.2 that encoded a new enzyme with two seine protease domains. Then, a PCR-based strategy was used to clone the full-length cDNA for this novel polyprotease. To this end, specific oligonucleotides derived from the genomic sequences were used to screen a panel of human cDNA libraries for transcripts corresponding to this new polyserine protease. The sequences of the designed primers were pol3-f1 (forward) 5'- GGCTGCCCTGCAGTTGCC-3', and pol3-r1 (reverse) 5'-CAGGTGGTGGTCAGTAGGG-3' for polyserase-3. All PCRs were performed in a GeneAmp 2400 PCR system (PerkinElmer Life Sciences) for 40 cycles of denaturation (94°C, 20 s), annealing (63°C, 20 s), and extension (68°C, 60 s). After cloning of the PCR-amplified products in pBlueScriptII (Invitrogen), their identities were confirmed by nucleotide sequencing using the kit DR terminator TaqFS and the automatic DNA sequencer ABI-PRISM 310 (Perkin-Elmer Life Sciences). Sequence analysis of the RACE-extended cDNA clones led us to complete the identification of the new polyserase. Finally, the full-length cDNA was obtained by PCR using the primers ATGpol3 (forward) 5'-ATGAAGTGGTG CTGGGGCCCA-3', ENDpol3 (reverse), 5'-TCAGCAGCTGGTTGGTTGGCT-3'. PCR conditions were as above, but with 280 s of extension. Nucleotide and protein sequence analysis were carried out using different programs available [35,40].
Nylon filters containing poly(A)+ RNAs of diverse human tissues were prehybridized at 42°C for 3 h in 50% formamide, 5× SSPE, 10× Denhardt's solution, 2% SDS, and 100 μg/ml of denatured herring sperm DNA. Hybridization was performed with a radiolabeled 628 pb EcoRI-BamHI fragment of polyserase-3. After hybridization for 20 h under the same conditions, filters were washed with 0.1× SSC, 0.1% SDS for 2 h at 50°C, and exposed to autoradiography.
Construction of expression vectors and purification of recombinant proteins
To analyze the expression of polyserase-3 in eukaryotic cells, the full-length cDNA encoding this protein was PCR-amplified and cloned between the HindIII and NotI sites of a modified pCEP4 expression vector (Invitrogen), which facilitated to add a HisTag tail at the C-terminus of the recombinant protein. The oligonucleotides used to do this were 5'-TTAAAGCTTATGAAGTGGTGCTGGGGCC-3' (forward) and 5'-TGTGCGGCCGCGCAGCTGGTTGGTTGGCTA-3' (reverse) where the HindIII and NotI sites are indicated in bold. A BssHII site was created in the coding sequence (positions 809 to 814) which allowed us to introduce a FLAG epitope between the two serine protease domains, using the oligonucleotides 5'-CGCGCGACTACAAGGACGACGATGACAAG-3' and 5'-CGCGCTTGTCATCGTCGTCCTTGTAGTCTG-3'. The resulting vector, pCEP-pol3, was transfected into HeLa and 293-EBNA cells using the LipofectAMINE reagent (Life Technologies, Inc.). When indicated, tunicamycin was added to the cells at a final concentration of 1 μg/mL. Expression of each independent serine protease domain as well as the entire protease in bacterial cells was carried out using the pGEX-2TK vector (Amersham Biosciences). To this end, the first serine protease domain was PCR-amplified from pCEP-pol3 using the oligonucleotides 5'-GAGGGCAACACAGTCCCTGGCGAG-3' (forward) and 5'-GTAGAGGCCCCAGAGACCCGA-3' (reverse), and cloned into the SmaI site of the pGEX vector to generate pGEX-pol3Spd1. Similarly, the second serine protease domain was amplified by PCR using the oligonucleotides 5'-TCGGGTCTCTGGGGCCTCTAC-3' (forward) and 5'-GTGATGGTGATGGTGATGTGC-3' (reverse) and cloned as above to prepare the construct pGEX-pol3Spd2. To produce the entire protease, a PCR-amplification was carried out with the oligonucleotides 5'-GAGGGCAACACAGTCCCTGGCGAG-3' and 5'-GTGATGGTGATGGTGATGTGC-3' and the PCR product was cloned as above to get pGEX-pol3 vector. A pGEX plasmid expressing ADAM23 disintegrin domain was used as control to assess quality in the further purification process of the fusion proteins, and as negative control of the serine protease activity in the enzymatic assays . Plasmids were transformed into BL21(DE3) pLysE Escherichia coli cells and expression was induced at 19°C using 0.4 mM of isopropyl-β-D-thiogalactoside (IPTG). After that, cells were collected, lysed and centrifuged, and the soluble fractions containing the recombinant proteins were purified as follows. A glutathione-Sepharose 4B (Amersham Biosciences) was initially used and the eluted proteins were subsequently loaded in a gel filtration column (Superdex 200, Amersham Biosciences). The quality of the purification process was followed by SDS-PAGE and Western blot analyses, using an anti-GST antibody (Amersham Biosciences). To evaluate the possibility that polyserase-3 forms dimers, the full-length cDNA was amplified using the oligonucleotides 5'-GCAAGATCTAACACAGTCCCTGGCGA GTGG-3' (forward) and 5'-TGCAAGCTTTCAGCAGCTGGTTGGTTGGCTTAT-3' (reverse), where the BglII and HindIII sites are indicated in bold. The PCR-product was digested with these restriction enzymes and cloned between these positions in pRSETB, which add a 6xHisTag tail at the N-terminus of the protein. The resulting vector (pRSETB-pol3) was transformed into BL21(DE3) pLysE. The production of the recombinant protein was as above; whereas the purification was carried out using a Ni-NTA column (Qiagen), and an anti 6xHisTag antibody (Qiagen) was used to detect the produced protein.
The catalytic activity of the recombinant proteins was analyzed using a panel of different proteins as potential substrates including type I collagen, type I gelatin, type I laminin, pro-uPA and fibrinogen. The assays were carried out with 5 μg of each protein in a buffer containing 50 mM Tris-HCl pH 7.4 and 150 mM NaCl, during 16 h at 37°C. The enzyme/substrate ratio (w/w) used in these experiments was 1/100. The resulting material was subjected to SDS-PAGE analysis. For inhibition assays, polyserase-3 was preincubated with AEBSF (0.1 mM), EDTA (2 mM), and E-64 (10 μM) for 30 min at 37°C, and then incubations were performed at the same conditions as above.
A three-dimensional model of each polyserase-3 serine protease domains was calculated using Swiss-Model, a semiautomated modeling server , and analyzed with the Swiss-Pdb Viewer. The amino acid sequence of each serine protease domain was compared with the sequences of the protein structures deposited in the Protein Data Bank. After analyzing structures of non-redundant proteins that had the highest structural quality and significant sequence similarity with each polyserase-3 catalytic domain, we chose the human β-tryptase (1a01), matriptase-1 (1eaxa) and human plasmin (1bmla) as templates. The templates were superimposed and aligned structurally. The quality of the resulting models was verified manually with Swiss-Pdb Viewer. The figures were rendered with POV-Ray .
After transfection, HeLa and 293-EBNA cells were fixed with 4% paraformaldehyde in PBS. Then, cells were permeabilized for 5 min with 0.2% Triton X-100 in PBS. Blocking was carried out with 15% fetal bovine serum in the same buffer. Blocked slices were incubated for 2 h with different dilutions of the primary anti-FLAG antibody, followed by 1 h incubation with a secondary fluorescein-conjugated goat anti-mouse antibody. Slides were coverslipped in the presence of Vectashield medium (Vector Laboratories) containing 4'-6'-diamidino-2-phenylindole hydrochloride (DAPI) and imaged by fluorescence microscopy.