mice than in PBS-treated mice (O.Melnyk, T.T., C.C., and M.S., unpublished data). Inhibition was not unexpected with ecotin M84R/M85R treatment, because uPA has been implicated in metastasis. However, wild-type ecotin is a poor, micromolar inhibitor of uPA; one interpretation of the data is that the decrease in tumor size and metastasis in the mouse model involves the inhibition of additional serine proteases. Thus, identification of the serine proteases expressed by PC-3 prostate cells may provide insight into the role of these proteases in cancer and prostate growth and development. In this report we have extended the strategy of using PCR with degenerate oligonucleotide primers that were designed by using conserved sequence homology (12–14) to identify additional serine proteases made by cancer cells. Five independent serine protease cDNAs derived from PC-3 mRNA were sequenced, including a novel serine protease, which we refer to as membrane-type serine protease 1 (MT-SP1), and the cloning and characterization of this cDNA that encodes a mosaic, transmembrane protease is reported.
Materials. All primers used were synthesized on a Applied Biosystems 391 DNA synthesizer. All restriction enzymes were purchased from New England Biolabs. Automated DNA sequencing was carried out on an Applied Biosystems 377 Prism sequencer, and manual DNA sequencing was carried out under standard conditions. N-terminal amino acid sequencing was performed on an ABI 477A by the University of California, San Francisco Biomolecular Resource Center. The synthetic substrates, Suc-AAPX-p-nitroanilide (pNA), [N-succinylalanyl-alanyl-prolyl-Xxx-pNA (Xxx=alanyl, aspartyl, glutamyl, phenylalanyl, leucinyl, methionyl, or arginyl)], and H-Arg-pNA, (arginyl-pNA), were purchased from Bachem. Deglycosylation was performed by using PNGase F (NEB, Beverly, MA). All other reagents were of the highest quality available and purchased from Sigma or Fisher unless otherwise noted.
Isolation of cDNA from PC-3 Cells. mRNA was isolated from PC-3 cells by using the polyATtract System 1000 kit (Promega). Reverse transcription was primed by using the “lock-docking” oligo(dT) primer (15). Superscript II reverse transcriptase (Life Technologies, Grand Island, NY) was used in accordance with the manufacturer’s instructions to synthesize the cDNA from the PC-3 mRNA.
Amplification of MT-SP1 Gene. The degenerate primers used for amplifying the protease domains were designed from the consensus sequences flanking the catalytic histidine (5′ His-primer) and the catalytic serine (3′ Ser-primer), similar to those described (12). The 5′ primer used is as follows: 5′-TGG (AG)TI (CAG)TI (AT)(GC)I GCI (GA)CI CA(CT) TG-3′, where nucleotides in parentheses represent equimolar mixtures and I represents deoxyinosine. This primer encodes at least the following amino acid sequence: W (I/V) (I/V/L/M) (S/T) A (A/T) H C. The 3′ primer used is as follows: 5′-IGG ICC ICC I(GC)(AT) (AG)TC ICC (CT)TI (GA)CA IG(ATC) (GA)TC-3′. The reverse complement of the 3′ primer encodes at least the following amino acid sequence: D (A/S/T) C (K/E/Q/H) G D S G G P.
Direct amplification of serine protease cDNA was not possible by using the above primers. Instead, the first PCR was performed with the 5′ His-primer and the oligo(dT) primer described above, by using the “touchdown” PCR protocol (16), with annealing temperatures decreasing from 52°C to 42°C over 22 rounds and 13 final rounds at 54°C annealing temperature. Cycle times were 1 min (denaturing), 1 min (annealing), and 2 min (extension) and were followed by one final extension time of 15 min after the final round of PCR. The template for the second PCR was 0.5 μl (total reaction volume 50 μL) of a 1:10 dilution of the first PCR mixture that was performed with the 5′ His-primer and the oligo(dT). The second PCR reaction was primed with the 5′ His- and the 3′ Ser-primers and performed by using the touchdown protocol described above. All PCRs used 12.5 pmol of primer for 50-μl reaction volume.
The product of the second reaction was purified on a 2% agarose gel, and all products between 400 and 550 bp were cut from the gel and extracted by using the QIAquick gel extraction kit (Qiagen, Chatsworth, CA). These products were digested with the BamHI restriction enzyme to cut any uPA cDNA, and all 400- to 500-bp fragments were repurified on a 2% agarose gel. These reaction products were subjected to a third PCR by using the 5′ His-primer and the 3′ Ser-primer by using the identical touchdown procedure. These reaction products were gel-purified and directly cloned into the pPCR2.1 vector by using the TOPO TA ligation kit (Invitrogen). DNA sequencing of the inserts determined the cDNA sequence from nucleotides 1,984 to 2,460 (see Fig. 1).
Northern Blot Analysis. 32P-labeled nucleotides were purchased from Amersham Pharmacia. A cDNA fragment containing nucleotides 1,173–2,510 was digested from expressed sequence tag w39209 by using restriction enzymes EcoRI and BsmbI, yielding a 1.3-kilobase nucleotide insert. Labeled cDNA probes were synthesized by using the Rediprime random primer labeling kit (Amersham Pharmacia) and 20 ng of the purified insert. Poly(A)+RNA membranes for Northern blotting were purchased from Origene (Rockville, MD; HB-1002, HB-1018) and CLONTECH (Human II 7759–1, Human Cancer Cell Line 7757). The blots were performed under stringent annealing conditions as described in ref. 17.
Construction of Expression Vectors. The mature protease domain and a small portion of the pro-domain (nucleotides 1,822–2,601) cDNA were amplified by using PCR from expressed sequence tag w39209 and ligated into the pQE30 vector (Qiagen). This construct is designed to overexpress the protease sequence from amino acids (aa) 596–855 with the following fusion: Met-Arg-Gly-Ser-His6-aa596–855. The Histag fusion allows affinity purification by using metal-chelate chromatography. The change from Ser-805, encoded by TCC, to Ala (GCT) was performed by using PCR. The presence of the correct Ser → Ala substitution in the pQE30 vector was verified by DNA sequence analysis.
Expression and Purification of the Protease Domain. The above-mentioned plasmids were separately transformed into Escherichia coli X-90 to afford high-level expression of recombinant protease gene products (18). Expression and purification of the recombinant enzyme from solubilized inclusion bodies was performed as described (19). Protein-containing fractions were pooled and dialyzed overnight at 4°C against 50 mM Tris (pH 8), 10% glycerol, 1 mM 2-mercaptoethanol, and 3 M urea. Autoactivation of the protease was monitored on dialysis against storage buffer (50 mM Tris, pH 8/10% glycerol) at 4°C by using the substrate Spectrozyme tPA (hexahydrotyrosyl-Gly-Arg-pNA, American Diagnostics, Greenwich, CT). Hydrolysis of Spectrozyme tPA was monitored at 405 nM for the formation of p-nitroaniline by using a Uvikon 860 spectrophotometer. Activated protease was bound to an immobilized p-aminobenzamidine resin (Pierce) that had been equilibrated with storage buffer. Bound protease was eluted with 100 mM benzamidine and the protein containing fractions were pooled. Excess benzamidine was removed by using FPLC with a Superdex 70 (Amersham Pharmacia) gel filtration column that was equilibrated with storage buffer. Protein containing fractions were pooled and stored at –80°C. The cleavage of the purified Ser805 Ala protease domain was performed at 37°C by addition of active recombinant protease domain to 10 nM. Cleavage was monitored by using SDS/ PAGE.
Determination of Substrate Kinetics. The purified serine protease domain was titrated with 4-methylumbelliferyl pguanidinobenzoate (MUGB) to obtain an accurate concen-