Portal de Periódicos da CAPES

Identification and Cloning of Human Placental Bikunin, a Novel Serine Protease Inhibitor Containing Two Kunitz Domains

1997; Elsevier BV; Volume: 272; Issue: 18 Linguagem: Inglês

10.1074/jbc.272.18.12202

1083-351X

Christopher W. Marlor, Katherine Delaria, Gary L. Davis, Daniel K. Muller, Jeffrey M. Greve, Paul P. Tamburini,

Protease and Inhibitor Mechanisms

Interrogation of the public expressed sequence tag (EST) data base with the sequence of preproaprotinin identified ESTs encoding two potential new members of the Kunitz family of serine protease inhibitors. Through reiterative interrogation, an EST contig was obtained, the consensus sequence from which encoded both of the novel Kunitz domains in a single open reading frame. This consensus sequence was used to direct the isolation of a full-length cDNA clone from a placental library. The resulting cDNA sequence predicted a 252-residue protein containing a putative NH2-terminal signal peptide followed sequentially by each of the two Kunitz domains within a 170-residue ectodomain, a putative transmembrane domain, and a 31-residue hydrophilic COOH terminus. The gene for this putative novel protein was mapped by use of a radiation hybrid panel to chromosome 19q13, and Northern analysis showed that the corresponding mRNA was expressed at high levels in human placenta and pancreas and at lower levels in brain, lung, and kidney. An endogenous soluble form of this protein, which was designated as placental bikunin, was highly purified from human placenta by sequential kallikrein-Sepharose affinity, gel filtration, and C18 reverse-phase chromatography. The natural protein exhibited the same NH2 terminus as predicted from the cloned cDNA and inhibited trypsin, plasma kallikrein, and plasmin with IC50 values in the nanomolar range. Interrogation of the public expressed sequence tag (EST) data base with the sequence of preproaprotinin identified ESTs encoding two potential new members of the Kunitz family of serine protease inhibitors. Through reiterative interrogation, an EST contig was obtained, the consensus sequence from which encoded both of the novel Kunitz domains in a single open reading frame. This consensus sequence was used to direct the isolation of a full-length cDNA clone from a placental library. The resulting cDNA sequence predicted a 252-residue protein containing a putative NH2-terminal signal peptide followed sequentially by each of the two Kunitz domains within a 170-residue ectodomain, a putative transmembrane domain, and a 31-residue hydrophilic COOH terminus. The gene for this putative novel protein was mapped by use of a radiation hybrid panel to chromosome 19q13, and Northern analysis showed that the corresponding mRNA was expressed at high levels in human placenta and pancreas and at lower levels in brain, lung, and kidney. An endogenous soluble form of this protein, which was designated as placental bikunin, was highly purified from human placenta by sequential kallikrein-Sepharose affinity, gel filtration, and C18 reverse-phase chromatography. The natural protein exhibited the same NH2 terminus as predicted from the cloned cDNA and inhibited trypsin, plasma kallikrein, and plasmin with IC50 values in the nanomolar range. The Kunitz (1Kunitz M. Northrop J.H. J. Gen. Physiol. 1936; 19: 991-1007Google Scholar, 2Laskowski M. Kato I. Annu. Rev. Biochem. 1980; 49: 593-626Scopus (1917) Google Scholar), Kazal (2Laskowski M. Kato I. Annu. Rev. Biochem. 1980; 49: 593-626Scopus (1917) Google Scholar), Serpin (3Potempa J. Korzus E. Travis J. J. Biol. Chem. 1994; 269: 15957-15960Google Scholar), and mucus (4Wiedow O. Schroeder J.-M. Gregory H. Young J.A. Christophers E. J. Biol. Chem. 1990; 265: 14791-14795Google Scholar) families of biological serine protease inhibitors play a vital role in the spatial and temporal regulation of in vivo proteolysis. The prototypical Kunitz inhibitor, bovine aprotinin (2Laskowski M. Kato I. Annu. Rev. Biochem. 1980; 49: 593-626Scopus (1917) Google Scholar), is a 58-amino acid protein containing three intrachain disulfide bonds in a spacing that is conserved in all family members (1Kunitz M. Northrop J.H. J. Gen. Physiol. 1936; 19: 991-1007Google Scholar, 2Laskowski M. Kato I. Annu. Rev. Biochem. 1980; 49: 593-626Scopus (1917) Google Scholar). Although the physiologic function of aprotinin is uncertain, it is a potent inhibitor of several serine proteases, and its potency against kallikrein and plasmin (5Fritz H. Wunderer G. Arzneimittel-Forshung. Drug Res. 1983; 33: 479-494Google Scholar) may be relevant to its clinical mode of action (5Fritz H. Wunderer G. Arzneimittel-Forshung. Drug Res. 1983; 33: 479-494Google Scholar, 6Davis R. Whittington R. Drugs. 1995; 49: 954-983Google Scholar), particularly in the reduction of perioperative blood loss. A human functional homolog of aprotinin has not been identified, although several larger human proteins containing one or more Kunitz domains are known. These include: tissue factor pathway inhibitor (TFPI), 1The abbreviations used are: TFPI, tissue factor pathway inhibitor; APP, amyloid precursor protein; EST(s), expressed sequence tag(s); dbEST, EST data base; PCR, polymerase chain reaction; bp, base pair(s); ORF, open reading frame; Tricine,N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; contig, group of overlapping clones. 1The abbreviations used are: TFPI, tissue factor pathway inhibitor; APP, amyloid precursor protein; EST(s), expressed sequence tag(s); dbEST, EST data base; PCR, polymerase chain reaction; bp, base pair(s); ORF, open reading frame; Tricine,N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; contig, group of overlapping clones. which contains three Kunitz domains (7Wun T.-Z. Kretzmer K.K. Girard T.J. Miletich J.P. Broze Jr., G.J. J. Biol. Chem. 1988; 263: 6001-6004Google Scholar) and inhibits both factor Xa and the factor VIIa-tissue factor complex (8Broze Jr., G.J. Blood Coagul. & Fibrinolysis. 1995; 6: S7-S13Google Scholar); TFPI-2 (9Sprecher C.A. Kisiel W. Mathewes S. Foster D.C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3353-3357Google Scholar), which contains two Kunitz domains (a bikunin) and is a potent inhibitor of the factor VIIa-tissue factor complex, factor XIa, and plasmin (10Petersen L.C. Sprecher C.A. Foster D.C. Blumberg H. Hamamoto T. Kisiel W. Biochemistry. 1996; 35: 266-272Google Scholar); and inter-α-trypsin inhibitor, a plasma-associated bikunin (11Kaumeyer J.F. Polazzi J.O. Kotick M.P. Nucleic Acids Res. 1986; 14: 7839-7850Google Scholar). In addition, the following proteins are known to contain a single Kunitz domain: COLα3/VI, the α(3) chain of type VI collagen (12Chu M.-L. Zhang R.-Z. Pan T.-c. Stokes D. Conway D. Kuo H.-J. Glanville R. Mayer U. Mann K. Deutzmann R. Timple R. EMBO J. 1990; 8: 385-393Google Scholar); HKI-B9, a human Kunitz inhibitor (13World Patent WO 93/14123Norris, N., Norris, K., Bjorn, S. E., Petersen, L. C., Foster, D. C., and Sprecher, C. A. (July 22, 1993) World Patent WO 93/14123.Google Scholar); the membrane-associated amyloid precursor proteins APP751(14Ponte P. Gonzalez-DeWhitt P. Schilling J. Miller J. Hsu D. Greenberg B. Davis K. Wallace W. Lieberburg I. Fuller F. Cordell B. Nature. 1988; 11: 525-527Google Scholar) and APP770 (15Tanaka S. Nakamura S. Ueda K. Kameyama M. Shiojiri S. Takahashi Y. Kitaguchi N. Ito H. Biochem. Biophys. Res. Commun. 1988; 157: 472-479Google Scholar); the amyloid precursor-like proteins (APLP) such as APLP2 (16Wasco W. Gurubhagavatula S. d. Paradis M. Romano D.M. Sisodia S.S. Hyman B.T. Neve R.L. Tanzi R.E. Nat. Genet. 1993; 5: 95-99Google Scholar). To identify novel human homologs of aprotinin we employed a bioinformatic approach that exploited the rapidly expanding human expressed sequence tags (ESTs) data base (17Lennon G.G. Auffray C. Polymeropoulos M. Soares M.B. Genomics. 1996; 33: 151-152Google Scholar,18Hillier L. Lennon G. Becker M. Bonaldo M.F. Chiapelli B. Chissoe S. Dietrich N. DuBuque T. Favello A. Gish W. Hawkins M. Hultman M. Kucaba T. Lacy M. Le M. Le N. Mardis E. Moore B. Morris M. Parsons J. Prange C. Rifkin L. Rohlfing T. Schellenberg K. Soares M.B. Tan F. Thierry-Meg J. Trevaskis E. Underwood K. Wohldman P. Waterson R. Wilson R. Marra M. Genome Res. 1996; 6: 807-828Google Scholar). This resulted in the discovery of a novel human gene product designated as placental bikunin. The full-length sequence of the bovine protein preproaprotinin 2Residues of the placental bikunin sequence and fragments thereof are numbered consecutively with positive integers in an NH2- to COOH-terminal direction with residue number 1 representing the first residue of the native protein following removal of the signal peptide. Amino acids within the signal peptide are numbered consecutively with negative integers in a COOH- to NH2-terminal direction with residue −1 representing the signal peptide residue adjacent to the bond hydrolyzed during signal peptide removal. (National Center for Biological Information (NCBI) sequence 162769) MKMSRLCLSVALLVLLGTLAASTPGCDTSNQAKAQRPDFCLEPPYTGPCKARIIRYFYNAKAGLCQTFVYGGCRAKRNNFKSAEDCMRTCGGAIGPWENL was used to query the data base of ESTs (dbEST) at the NCBI using the tBLASTn algorithm (19Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Google Scholar). This yielded cDNA sequences that when translated, encoded proteins with a cysteine spacing that was similar (R35464) or identical (R74593) to the spacing characteristic of the Kunitz family of serine protease inhibitor domains, but which were clearly different in their overall sequence from known human Kunitz family members. The nucleotide sequences of these ESTs were then used to re-query dbEST using BLASTn (19Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Google Scholar) and FASTA (20Pearson W.R. Lipman D.J. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 2444-2448Google Scholar) to generate overlapping nucleotide sequences that extended the sequence of the putative cDNA. ESTs that overlapped these two sequences were aligned using AssemblyLIGN (Ref. 21Program Manual for the Wisconsin Package (1994) Version 8, Genetics Computer Group, Madison, WI.Google Scholar, Oxford Molecular Group, Campbell, CA). This process was repeated by searching dbEST with the newly identified sequences until the overlapping ESTs extended from a putative ATG initiation site to a poly(A) at the 3′ end. Alignment of the sequences was used to assign the consensus base at each position within the alignment while giving additional weight to those bases defined as being in a region of high quality sequence. To obtain a cDNA encoding the entire extracellular region of the bikunin, the EST alignments were used to design a 5′ PCR primer, CACCTGATCGCGAGACCCC, based on the sequence of EST R34808 and a 3′ primer, CGAAGCTTCATCTCCGAAGCTCCAGACG (containing a HindIII site), based on the sequence of EST R74593. A 30-cycle PCR (95 °C for 5 min, 1 cycle; 95 °C for 1 min, 55 °C for 30 s, 72 °C for 2 min, 30 cycles; 72 °C for 5 min, 1 cycle) using a GeneAmp PCR reagent kit (Perkin-Elmer) amplified a 780-bp fragment from human placental cDNA (CLONTECH), which was then ligated into the pCRII vector (Invitrogen, San Diego). This clone was sequenced (FDA submission grade) on both strands by LARK Sequencing Technologies (Houston) to confirm the presence of an open reading frame (ORF) related to the EST consensus sequence. The PCR-derived cDNA fragment was then used to probe a human placental cDNA library (Unizap XR library, Stratagene, LaJolla, CA). Briefly, 2 × 106 λ plaques were plated on XL1 Blue cells onto NZY plates (Northeast Laboratory, Waterville, ME) at 37 °C, overnight. Plaques were transferred to nitrocellulose, denatured in 1.5m NaCl, 0.5 m NaOH for 2 min; neutralized in 1.5 m NaCl, 0.5 m Tris (pH 8.0) for 5 min; rinsed in 0.2 m Tris (pH 7.5), 2 × SSC (15 mm sodium citrate (pH 7.6) containing 150 mmNaCl), then blotted onto Whatman 3MM paper and cross-linked by UV irradiation with a Stratalinker (Stratagene). Filters were then prehybridized at 42 °C for 3 h in 50% formamide containing 5 × SSPE (10 mm NaH2PO4 (pH 7.4), containing 1 mm EDTA and 150 mm NaCl), 5 × Denhardt's reagent, and 0.1% SDS. The 780-bp PCR fragment was liberated from the pCRII plasmid by EcoRI (New England Biolabs, Beverly, MA) digestion, gel purified on a 1% agarose gel, and eluted with a gel extraction kit (Qiagen, Chatsworth, CA). Approximately 40 ng of purified fragment was heat denatured (100 °C for 5 min) and labeled with [32P]dCTP (Amersham Corp.) with High Prime labeling reagent (Boehringer Mannheim) using the random priming method. Unincorporated label was removed using Biospin columns (Bio-Rad). The probe was again heat denatured as described above, added to the filters, and hybridized at 42 °C overnight. Filters were then washed twice for 10 min in 2 × SSC, 0.5% SDS at 25 °C, followed by two 30-min washes in 1 × SSC, 0.1% SDS at 65 °C, and exposed to Kodak XAR film overnight at −80 °C with an intensifying screen. After three rounds of screening and plaque purification, five independent clones were isolated. In vivoexcision in SOLR cells and DNA preparation were performed according to the manufacturer's instructions (Qiagen). DNA was sequenced on both strands by the dideoxynucleotide termination method (22Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Google Scholar) at the sequencing facility at Yale University (New Haven, CT). A human multiple tissue Northern blot (CLONTECH) containing approximately 2 μg of poly(A)+ RNA/lane was probed with the 780-bp bikunin PCR fragment that was labeled with [32P]dCTP as described above. Hybridization and washing conditions were according to the manufacturer's instructions. The chromosomal location of the gene encoding placental bikunin was determined by PCR amplification in conjunction with the Stanford G3 hybridization panel (Research Genetics, Huntsville, AL). The following primers based on the cDNA sequence of placental bikunin were designed: sense, ATCCACGACTTCTGCCTGGTGTCG; antisense, GACAGTGGCACATTTCTTGAGG. These primers were used to amplify a 174-bp nucleotide fragment of human genomic DNA encoding a portion of the NH2-terminal Kunitz domain. Amplification was achieved with the following conditions: 95 °C for 10 min (cycle 1); 95 °C for 30 min, 55 °C for 15 min, 72 °C for 30 min, (cycles 2–41); 72 °C for 5 min (cycle 42). Data were submitted to the Stanford RH Mapping Center and analyzed using the program RHMAP. A whole frozen human placenta (Analytical Biological Services, Inc. Wilmington, DE), weighing between 0.7 and 0.8 kg, was thawed to 4 °C, cut into 0.5–1.0-cm pieces on ice, and then washed with 600 ml of phosphate-buffered saline. Tissue (240 g, wet weight, per run) was added to 300 ml of 0.1 m Tris-HCl (pH 8.0) containing 0.1m NaCl (buffer A), then homogenized in a Waring blender (maximum speed for 2 min). This procedure was repeated until all the tissue was processed. The supernatant, after centrifugation of this homogenate at 4,500 × g for 60 min at 4 °C, was collected, filtered through cheesecloth, and then applied to a kallikrein-Sepharose affinity column that had been equilibrated in buffer A at 4 °C. This column was made by attaching 70 mg of bovine pancreatic kallikrein to 5.0 ml of CNBr-activated Sepharose (Pharmacia Biotech Inc.) according to the manufacturer's instructions. After loading, the column was washed with buffer A until the absorbance at 280 nm of the eluent decreased to zero. The column was further washed with buffer A containing 0.5 m NaCl and then eluted with 3 volumes of 0.2 m acetic acid (pH 4.0). The flow rate was 2 ml/min throughout. Fractions containing kallikrein and trypsin inhibitory activity were pooled, frozen, and concentrated by lyophilization. The lyophilized sample was reconstituted in 1.0 ml of 0.1 m Tris-HCl (pH 8.0), containing 0.15 mNaCl, and 0.01% Triton X-100 and applied in 200-μl aliquots to a Superdex 75 10/30 column (Pharmacia) equilibrated and eluted (0.5 ml/min) with buffer A at room temperature. Fractions (0.5 ml) containing resolved peaks of trypsin or kallikrein inhibitory activity (see below) were pooled separately, adjusted to pH 2.5 by the addition of trifluoroacetic acid, then applied directly to a Vydac C18 reverse-phase column (5 μm, 0.46 × 25 cm) which had been equilibrated with 20% (v/v) acetonitrile in 0.1% (w/v) trifluoroacetic acid. Following a 20-ml wash with equilibration buffer, the column was eluted with a linear gradient of 20–80% acetonitrile in 0.1% trifluoroacetic acid over 50 min. The flow rate was 1 ml/min throughout. Fractions (1.0 ml) containing trypsin and kallikrein inhibitory activity were pooled, concentrated using a Speed-Vac concentrator (Savant Instruments, Farmingdale, NY), and stored at −20 °C until needed. The purification of placental bikunin was monitored with assays for the in vitroinhibition of bovine trypsin and human plasma kallikrein. To monitor trypsin inhibition, column fractions were preincubated with bovine trypsin (Sigma) for 10 min, after which reactions were initiated at 25 °C by the addition of 50 μl of Tos-G-P-K-AMC substrate (Bachem Bioscience Inc., King of Prussia, PA) to achieve the following final component concentrations in a 0.17-ml reaction volume: trypsin (17.5 μg/ml); purification fraction (20 μl), Tos-G-P-K-AMC (33 μm) in 50 mm Hepes buffer (pH 7.5) containing 0.1 m NaCl, 2.0 mm CaCl2, 0.01% (v/v) Triton X-100 (buffer B). To monitor plasma kallikrein inhibition, aliquots (20 μl) of column samples were preincubated for 15 min at 25 °C with 7.0 nm final plasma kallikrein (Enzyme Research Laboratories, South Lafayette, IN) in 50 mmTris-HCl (pH 8.0), containing 50 mm NaCl and 0.01% Triton X-100 (buffer C). Reactions were then initiated with 66 μm final P-F-R-AMC (Sigma). Real time formation of coumarin was determined fluorometrically (excitation = 370 nm, emission = 432 nm) in 96-well microtiter plates (Perkin-Elmer) on a Perkin Elmer LS-50B fluorometer equipped with a plate reader. To determine IC50 values, active site concentrations of trypsin, plasma kallikrein, and plasmin were determined by titration with p-nitrophenyl p′-guanidinobenzoate HCl (Sigma), as described (23Chase T. Shaw E. Methods Enzymol. 1970; 19: 20-27Google Scholar). Natural placental bikunin was quantified by titration against 2.2 nm bovine trypsin. Enzyme and inhibitor were mixed in a total volume of 990 μl of the appropriate buffer (see below) and incubated for 5 min at 37 °C. Reactions were initiated by the addition of substrate. Initial component concentrations were as follows: bovine trypsin activity, buffer B with [E 0] = 50 pm, inhibitor (eight concentrations in the range 0–0.8 nm) and 30 μm Tos-G-P-K-AMC substrate (K m = 29 μm); human plasmin (American Diagnostica, Inc., Greenwich, CT), 50 mm Tris-HCl (pH 7.5), 0.1 mNaCl, and 0.02% Triton X-100 with [E 0] = 50 pm, inhibitor (six concentrations in the range 0–3 nm) and 500 μm Tos-G-P-K-AMC (K m = 726 μm); human plasma kallikrein, buffer B with [E 0] = 2.5 nm, inhibitor (eight concentrations in the range 0–12 nm) and 100 μm P-F-R-AMC (K m = 457 μm). Hydrolysis of AMC-conjugated peptides was monitored on a Perkin-Elmer model LS50B fluorometer (excitation = 370 nm, emission = 432 nm) over a 2-min period. Percent inhibition (%I) values were determined from the equation: %I = 100 × [1-F 0/F 1], whereF 0 and F 1 are, respectively, the fluorescence in the presence and absence of inhibitor. NH2-terminal sequencing was performed on a Hewlett-Packard model G1005A protein sequencing system using Edman degradation and Version 3.0 sequencing methods as per the manufacturer's instruction (24Miller C.G. Methods: Companion Methods Enzymol. 1994; 6: 315-333Google Scholar). Samples were loaded onto the miniature biphasic reaction column and washed with 1 ml of 2% trifluoroacetic acid prior to the initiation of Edman chemistry. SDS-polyacrylamide gel electrophoresis was performed using 10–20% Tricine-buffered polyacrylamide gels (Novex, San Diego, CA) according to the manufacturer's specifications and developed by silver staining with a Daiichi Silver Stain-II kit (Integrated Separation Systems, Natick, MA). To identify novel human EST sequences with homology to the Kunitz family of serine protease inhibitors, the dbEST (17Lennon G.G. Auffray C. Polymeropoulos M. Soares M.B. Genomics. 1996; 33: 151-152Google Scholar, 18Hillier L. Lennon G. Becker M. Bonaldo M.F. Chiapelli B. Chissoe S. Dietrich N. DuBuque T. Favello A. Gish W. Hawkins M. Hultman M. Kucaba T. Lacy M. Le M. Le N. Mardis E. Moore B. Morris M. Parsons J. Prange C. Rifkin L. Rohlfing T. Schellenberg K. Soares M.B. Tan F. Thierry-Meg J. Trevaskis E. Underwood K. Wohldman P. Waterson R. Wilson R. Marra M. Genome Res. 1996; 6: 807-828Google Scholar) was queried with the protein sequence of preproaprotinin using the tBLASTn algorithm (19Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Google Scholar). Initially, two ESTs from human placenta (accession numbers R35464 and R74593) were identified. Translation of R74593yielded a theoretical ORF of 108 residues which was flanked by stop codons and contained six cysteine residues in a spacing characteristic of Kunitz domains. A 110-residue ORF within R35464 also contained a Kunitz-like protein sequence; however, the second cysteine from the NH2-terminal end was replaced by a phenylalanine. Reinterrogation of dbEST with the nucleotide sequences of R35464 andR74593 established that the 3′ nucleotide sequence flanking the Kunitz domain encoded in R35464 was identical to the 5′ sequence flanking the Kunitz domain encoded in R74593. This suggested that the two ESTs were each part of a common cDNA that encoding a bikunin. Several overlapping ESTs were obtained upon reinterrogation with R35464 andR74593, which were in turn used to reinterrogate dbEST. In this iterative fashion, a large number of overlapping ESTs were obtained which could be aligned into an approximately 1,700-bp EST contig (Fig.1 A). Most of the ESTs comprising the contig were of placental origin, although some were recovered from infant and adult brain, breast, retina, olfactory epithelium, and ovary. A minimum of four ESTs overlapped each position within the contig except for the region between bp 800 and 900. This enabled the derivation of a consensus sequence from which at least some sequencing or cloning errors could be deconvoluted. For example, the stop codons flanking the Kunitz domain in EST R74593 were not evident in the consensus sequence, and the Kunitz-like sequence corresponding to that observed in ESTR35464 contained the full complement of six correctly spaced cysteine residues. The consensus nucleotide sequence was 1.6 kilobases in length (Fig. 1 B), extending to the start of a poly(A) tail, and encoded a 5′ ATG start site that was followed by a putative 248 ORF encoding the two Kunitz domains. Nucleotide primers based on specific EST sequences (located in Fig.1 B) were used to amplify a cDNA fragment from human placental cDNA with same size (780 bp) as predicted from the EST contig. The PCR-derived fragment (not shown) encoded an ATG start site followed by a 240-residue open reading frame which was identical to the EST consensus over a 222-residue stretch. Northern blot analysis using this PCR fragment as a probe revealed high levels of expression of a hybridizing mRNA in placenta and pancreas, with lower levels in kidney, lung, brain, and heart and undetectable levels in skeletal muscle (Fig. 2). The size of the mRNA (approximately 1.9 kilobases) was in reasonable agreement with that predicted by the EST contig (Fig. 1). This novel ORF was designated placental bikunin in accordance with its tissue abundance and sequence homology. The PCR fragment was used to probe a placental cDNA library, resulting in the isolation of a full-length placental bikunin cDNA (Fig. 3 A). The translated ORF within the full-length clone contained 252 amino acids and was identical to the EST consensus sequence over all but the first 15 residues. Analysis of the translated sequence with the program SigCleave identified the first 27 amino acid residues following the ATG start site (defined herein as residues −1 to −27) as a likely signal peptide (Fig. 3 A). This was followed by a 225-amino acid mature protein sequence beginning with the sequence ADRER- and containing two tandem Kunitz-like inhibitor domains within residues 7–64 and 102–159, respectively. A 24-residue hydrophobic segment between residues 171 and 194 of this mature protein sequence was followed by a hydrophilic tail of 31 residues. Analysis of the region surrounding the hydrophobic segment using the method of Kyte and Doolittle (25Kyte J. Doolittle R.F. J. Mol. Biol. 1982; 157: 105-132Google Scholar, 26Jahnig F. Fasman G.D. Prediction of Protein Structure and the Principles of Protein Conformation. Plenum Press, New York1989: 707-717Google Scholar) highlighted the region as a probable membrane anchor sequence. Several proline residues were evident immediately NH2-terminal to the putative transmembrane domain. Two potential N-linked glycosylation sites characterized by NXS/T motifs were evident at position 30 within the first Kunitz domain and at position 67 within the 38-amino acid segment separating the two Kunitz domains. These features are depicted schematically in Fig. 3 B. The entire nucleotide sequence of placental bikunin (Fig.3 A) was searched using BLAST against the following data bases: GenBank (current to 9/20/96), EMBL (release 47.0), GeneSeq DNA and protein (release 20.0), PIR (release 49.0), and PatchX (release 49.0). No significant homologies or identities to the coding sequence were observed. However, a portion of the 3′-untranslated sequence in the reverse direction was identical to a cDNA fragment found to be expressed differentially in pancreatic cancer (accession numberZ36849). Alignment of the Kunitz domains of placental bikunin with other Kunitz domains showed that they contained the characteristic conserved spacing of the six conserved cysteine residues as well as the conserved ..FXYXGCXGNXN.. motif surrounding the fourth cysteine residue (Fig. 4). Although the Kunitz domains within placental bikunin fragments 7–64 and 102–159 are clearly novel family members, they exhibited percent identities with other human Kunitz domains which ranged between 29 and 50%. The amino acid residue COOH-terminal to the second cysteine residue from the NH2 terminus strongly influences the protease specificity of Kunitz domains (2Laskowski M. Kato I. Annu. Rev. Biochem. 1980; 49: 593-626Scopus (1917) Google Scholar). In each of the Kunitz domains of placental bikunin this position is occupied by an arginine residue, suggesting that the domains may have specificity toward inhibition of trypsin-like serine proteases (2Laskowski M. Kato I. Annu. Rev. Biochem. 1980; 49: 593-626Scopus (1917) Google Scholar). The chromosomal location of the gene encoding placental bikunin was determined using primers based on the cDNA sequence encoding the full-length protein. Accordingly, the closest linkage marker to the fragment amplified from a Stanford G3 radiation hybrid panel was identified as D19S228 on chromosome 19 (19q13). This analysis generated an LOD score of 12.68. This agrees with the finding that 13 of the 29 ESTs used in the construction of our contig map have been mapped independently within the UniGene set (27Boguski M.S. Schuler G.D. Nat. Genet. 1995; 10: 369-371Google Scholar) to an unidentified transcript close to this region between D19S224 and D19S408. These ESTs include:N40851, H16866, R34808, N57450, R34701, H39840, H95233, H39841, N30199,N29508, N26910, H16757, and N27732. To characterize this novel serine protease inhibitor, we purified the naturally occurring protein from human tissue. Placenta was selected as the source material for purification because of the relatively high levels of placental bikunin mRNA expression in this tissue. The possibility that placental bikunin might bind tightly to trypsin-like serine proteases was exploited in these efforts through implementation of affinity chromatography over a column of immobilized kallikrein as the initial purification step following tissue homogenization. Accordingly, a small fraction of the total trypsin and kallikrein inhibitor activity present in the soluble fraction of a placental extract bound this column and could be recovered subsequently by elution at acidic pH. Gel filtration chromatography of this sample yielded a peak of kallikrein and trypsin inhibitory activity which eluted with an apparent molecular mass between 15 and 40 kDa based on calibration of the column with molecular mass standards under identical conditions (Fig. 5 A). Reverse-phase C18 chromatography (Fig. 5 B) yielded four peaks of inhibitory activity. The first peak of activity (peak 1) had the largest number of units of activity, yet had the least amount of protein based on its A215 nm, whereas peak 2 contained TFPI-2, as determined by NH2-terminal sequence analysis. SDS-polyacrylamide gel electrophoresis analysis of the fraction containing peak 1, followed by silver staining (Fig. 6), yielded a single major band with an apparent molecular mass of 24 kDa. A summary of the purification of the native protein is shown in TableI. Although determination of the fold purification and yield was complicated by the presence of other protease inhibitors in the crude homogenate, the purification scheme achieved at least a 4,700-fold purification of the inhibitor based on the specific activity of the final preparation relative to the starting homogenate.Figure 6Reducing SDS-polyacrylamide gel electrophoresis of a novel serine protease inhibitor purified from human placenta. Molecular size markers (lane 2) included, from top to bottom: ovalbumin, 45 kDa; carbonic anhydrase, 29 kDa; β-lactoglobulin, 18.4 kDa; lysozyme, 14.3 kDa; bovine trypsin inhibitor, 6.2 kDa; and insulin, 3 kDa. Purified placental serine protease inhibitor (0.5 μg) was applied tolane 1. The Tricine-SDS-polyacrylamide gel was silver stained as described under “Experimental Procedures.”View Large Image Figure ViewerDownload (PPT)Table IPurification of a novel serine protease inhibitor from human placentaStepVolumeTotal ProteinUnitsaOne unit is the amount of inhibitor that inhibits 50% of trypsin activity assayed according to “Experimental Procedures.”Units/mgmlmgPlacental supernatant1,800.075,0601-bDetermined from the absorbance at 280 nm.3,000,00040.0Kallikrein affinity20.03.361-bDetermined from the absorbance at 280 nm.16,0004,880Superdex 7515.00.13bDetermined from the absorbance at 280 nm.3,19124,546C181.00.0005cDetermined by active site titration with trypsin under stoichiometric conditions.91189,580a One unit is the amount of inhibitor that inhibits 50% of trypsin activity assayed according to “Experimental Procedures.”b Determined from the absorbance at 280 nm.c Determined by active site titration with trypsin under stoichiometric conditions. Open table in a new tab NH2-terminal sequence analysis of the final preparation (Fig. 7) yielded an amino acid sequence that was identical over the entire 50 cycles analyzed, to the NH2terminus of mature placental bikunin as predicted by a SigCleave analysis of the translated full-length cDNA (Fig. 3 A). High quality sequence extended well into the NH2-terminal Kunitz domain. The cysteine residues within this sequence were not detected because cysteine is recovered in low yield from the sequencer. Interestingly, the asparagine at amino acid residue 30 of the sequence was also not detected, as would be expected if it were glycosylated as predicted. Residues 35 and 48 could not be determined presumably because serine is recovered in low yield. The only other detectable NH2-terminal sequence within the purified sample was identical to the main sequence except that it commenced at residue 6 and represented only 6% of the total material sequenced. Peaks 3 and 4 recovered from C18 reverse-phase chromatography (Fig.5 B) remain unidentified. Similar efforts to isolate placental bikunin from the membrane fraction following solubilization with 1% (v/v) Triton X-100 did not yield detectable amounts of the inhibitor (data not shown). As might be predicted from the fact that placental bikunin contains Kunitz inhibitor domains, the natural form of the protein was a potent inhibitor of the following serine proteases: bovine trypsin (IC50 = 0.35 nm), human plasmin (IC50 = 2.5 nm), and human plasma kallikrein (IC50 = 8.0 nm). We have described the identification of a novel human gene product containing two Kunitz inhibitor domains. This was designated as placental bikunin based on its sequence homology and the tissue abundance of its mRNA. The discovery of this protein was made possible through interrogation of the dbEST with preproaprotinin followed by implementation of an EST “walk” to establish a theoretical ORF. This ORF was used to direct the cloning of a corresponding full-length placental cDNA that encoded the protein. The gene encoding placental bikunin was assigned to chromosome 19q13, where it mapped adjacent to D19S228. Evidence that the placental bikunin gene directs the expression of a protein was obtained from the isolation of a functional serine protease inhibitor from human placenta which possessed the same NH2 terminus as predicted from the placental bikunin cDNA. The isolated cDNA for placental bikunin encodes a protein that contains a signal peptide, a hydrophobic segment COOH-terminal to the two Kunitz domains, and a COOH-terminal hydrophilic tail. This suggests that the mature full-length protein encoded by the cDNA is targeted to the Golgi compartment following synthesis and may exist as a transmembrane protein. In this scenario, the 170-amino acid Kunitz-containing fragment would be exposed either to the extracellular milieu or lumen of a vesicular compartment depending on its trafficking pathway. Accordingly, NH2-terminal sequence analysis verified that the natural protein had undergone removal of the signal peptide predicted by the cDNA and that it had therefore likely been processed through the secretory pathway. The significance of the finding that the natural protein was recovered in the soluble fraction of placental homogenates without using detergents is open to speculation. Either the hydrophobic segment was present in the natural protein but did not act as a transmembrane domain, or it was absent from the protein as a consequence of alternate splicing or proteolytic processing. Proteolytic processing could have occurred physiologically as has been described for other Kunitz-containing proteins with transmembrane segments such as APP751 and APP770 (14Ponte P. Gonzalez-DeWhitt P. Schilling J. Miller J. Hsu D. Greenberg B. Davis K. Wallace W. Lieberburg I. Fuller F. Cordell B. Nature. 1988; 11: 525-527Google Scholar, 15Tanaka S. Nakamura S. Ueda K. Kameyama M. Shiojiri S. Takahashi Y. Kitaguchi N. Ito H. Biochem. Biophys. Res. Commun. 1988; 157: 472-479Google Scholar) or as a consequence of tissue homogenization. Although the size of the natural protein was consistent with that predicted from the ORF within the cloned cDNA, it is unclear how much of the protein sequence encoded by the cDNA is actually present in the purified natural protein. The presence of the NH2-terminal Kunitz domain was clearly evident by NH2-terminal sequencing, and this domain alone could have accounted for the protease inhibitor activity of the natural protein. Because efforts to elucidate the entire covalent structure of the protein have been hampered by the small amounts of purified protein, we have resorted to an immunoblot evaluation of the presence of the predicted domains within the native protein. Preliminary results using antibodies directed against the NH2- and COOH-terminal Kunitz domains (not shown) indicate that only the NH2-terminal domain is present in the 24-kDa species following purification, whereas the antibody against the COOH-terminal domain reacted with a band at 6 kDa in the same preparation, indicating that the COOH-terminal Kunitz domain was present but not covalently attached to the larger protein. Although placental bikunin seems to represent a low abundance protein as judged by its low recovery from human placenta, it is possible that the isolation procedure selected out a subpopulation of the inhibitor. For example, affinity chromatography over an immobilized protease would not have purified inhibitor that was bound to endogenous protease either prior to or as a consequence of tissue homogenization. The physiological function of placental bikunin is a matter of speculation at present. Its potential for existence extracellularly perhaps in part as a cell surface-associated protein raises the possibility that the protein could play a role in the regulation of extracellular proteolytic cascades that are activated in close proximity to cell membranes. Based on the potency of the natural protein against plasma kallikrein, such pathways could include the sequelae of contact activation triggered by vascular injury, which include the processes of kinin formation from high molecular weight kininogen, coagulation via the intrinsic pathway, and complement activation. On the other hand, the protein could function in the regulation of fibrinolysis based on its potency against plasmin. The elucidation of the biological functions of placental bikunin will require further study of its post-translational processing, subcellular distribution, cellular expression pattern, and protease specificity. We acknowledge the following members of the research staff at the Bayer Research Center for important contributions: Carla Pellegrino (protein sequencing), John Kupcho (amino acid analysis), and David Murray and Rathin Das (Northern blot analysis).