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

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 with a were then at °C for in containing 5 × mm (pH containing 1 mm and 150 mm 5 × and The 780-bp PCR fragment was from the pCRII by gel purified on a and with a gel kit CA). of purified fragment was denatured °C for 5 and with with reagent using the was using The probe was denatured as to the and at °C overnight. were then for in 2 × at °C, followed by two in 1 × at °C, and to at °C with an three of and were In in cells and were to the was sequenced on both strands by the F. S. Proc. Natl. Acad. Sci. U. S. A. Scholar) at the at A human tissue Northern containing 2 of was with the 780-bp bikunin PCR fragment that was with as and were to the The of the gene encoding placental bikunin was by PCR in with the panel The following based on the cDNA sequence of placental bikunin were These were used to a nucleotide fragment of human encoding a of the NH2-terminal Kunitz was with the following 95 °C for 95 °C for 30 min, 55 °C for min, 72 °C for 30 min, 72 °C for 5 were to the Center and using the A human placenta Biological and was to °C, into on and then with of was to of m (pH 8.0) containing then in a for 2 This was repeated until all the tissue was The of this at × for at °C, was and then to a kallikrein-Sepharose that been in A at This was by of bovine kallikrein to of to the the was with A until the at of the to The was with A containing 0.5 m and then with of 0.2 m acid (pH The was 2 containing kallikrein and were and by The was in of m (pH containing and and in to a and with A at containing of or kallikrein were to by the of then to a C18 reverse-phase × which been with in a with the was with a of in acid The was 1 containing and kallikrein were using a and at °C until The of placental bikunin was with for the in of bovine and human plasma To were with bovine for min, which were at °C by the of of of to the following in a in mm (pH containing m NaCl, mm To plasma kallikrein of were for at °C with plasma kallikrein in (pH containing mm and were then with of was in plates (Perkin-Elmer) on a with a To IC50 site of trypsin, plasma kallikrein, and plasmin were by with as T. E. 19: Scholar). placental bikunin was by against bovine and inhibitor were in a of of the and for 5 at 37 were by the of were as bovine with inhibitor in the and 30 m human plasmin mm (pH 7.5), and with inhibitor in the and m human plasma kallikrein, with inhibitor in the and m of was on a a values were from the × and 1 the in the presence and of NH2-terminal was on a protein using and Version as the 1994; 6: Scholar). were onto the and with 1 of acid to the initiation of gel was using San to the and by with a kit To identify novel human EST sequences with to the Kunitz family of serine protease the dbEST (17Lennon G.G. Auffray C. Polymeropoulos M. Soares M.B. Genomics. 1996; 33: 151-152Google Scholar, 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 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). two ESTs from human placenta and were of a of which was by and cysteine in a spacing characteristic of Kunitz A within a protein the cysteine from the NH2-terminal was by a of dbEST with the nucleotide sequences of that the 3′ nucleotide sequence the Kunitz encoded in was identical to the 5′ sequence the Kunitz encoded in R74593. This that the two ESTs were each of a cDNA that encoding a bikunin. overlapping ESTs were with which were in used to In this a number of overlapping ESTs were which be aligned into an EST contig of the ESTs the contig were of placental although were from and brain, and A of ESTs overlapped each position within the contig for the region and This the of a consensus sequence from which at or be the the Kunitz in EST were not in the consensus and the sequence corresponding to that in the of cysteine The consensus nucleotide sequence was in 1 to the of a poly(A) and encoded a 5′ ATG site that was followed by a putative encoding the two Kunitz based on EST sequences in were used to a cDNA fragment from human placental cDNA with same as predicted from the EST The PCR-derived fragment encoded an ATG site followed by a open reading frame which was identical to the EST consensus a Northern analysis using this PCR fragment as a probe high levels of of a mRNA in placenta and with lower levels in lung, brain, and and levels in The of the mRNA was in with that predicted by the EST contig This novel was designated placental bikunin in with its tissue and sequence The PCR fragment was used to probe a placental cDNA library, resulting in the isolation of a full-length placental bikunin cDNA The within the full-length clone acids and was identical to the EST consensus sequence all but the first of the sequence with the identified the first acid following the ATG site as −1 to as a signal peptide This was followed by a acid protein sequence with the sequence and containing two inhibitor domains within and A and of this protein sequence was followed by a hydrophilic of of the region the using the of and J. J. Mol. Biol. 157: Scholar, F. of and the of Scholar) the region as a sequence. were NH2-terminal to the putative transmembrane potential by were at position 30 within the first Kunitz and at position within the acid the two Kunitz These are in B. The entire nucleotide sequence of placental bikunin was using against the following data to and protein and or to the sequence were a of the sequence in the direction was identical to a cDNA fragment to be expressed in Alignment of the Kunitz domains of placental bikunin with Kunitz domains showed that the characteristic conserved spacing of the conserved cysteine as as the conserved the cysteine residue Although the Kunitz domains within placental bikunin fragments and are clearly novel family exhibited with human Kunitz domains which and The acid residue COOH-terminal to the cysteine residue from the NH2 terminus the protease 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 by an that the domains may of serine (2Laskowski M. Kato I. Annu. Rev. Biochem. 1980; 49: 593-626Scopus (1917) Google Scholar). The of the gene encoding placental bikunin was using based on the cDNA sequence encoding the full-length the to the fragment amplified from a radiation hybrid panel was identified as on chromosome This analysis an of This with the that of the ESTs used in the of contig been mapped within the Nat. Genet. 1995; Scholar) to an to this region and These ESTs and To this novel serine protease inhibitor, we purified the protein from human was as the for of the high levels of placental bikunin mRNA in this The that placental bikunin to serine was exploited in these of a of kallikrein as the following tissue a of the and kallikrein inhibitor in the soluble of a placental this and be by at of this yielded a of kallikrein and which with an and based on of the with identical 5 C18 5 yielded of The first of the number of of the of protein based on its 2 as by NH2-terminal sequence gel analysis of the containing followed by yielded a single with an of A of the of the native protein is in Although of the and was by the presence of protease inhibitors in the the at a of the inhibitor based on the of the to the gel of a novel serine protease inhibitor purified from human Molecular from to bovine inhibitor, and placental serine protease inhibitor was The gel was as of a novel serine protease inhibitor from human is the of inhibitor that inhibits of to from the at from the at from the at by site with is the of inhibitor that inhibits of to from the at by site with in a new NH2-terminal sequence analysis of the yielded an acid sequence that was identical the entire to the of placental bikunin as predicted by a analysis of the full-length cDNA quality sequence extended into the NH2-terminal Kunitz The cysteine within this sequence were not cysteine is in from the the at acid residue 30 of the sequence was not as be it were as and not be serine is in The NH2-terminal sequence within the purified was identical to the sequence that it at residue and of the and from C18 reverse-phase to placental bikunin from the following with not of the inhibitor not be predicted from the that placental bikunin contains Kunitz inhibitor domains, the natural form of the protein was a potent inhibitor of the following serine bovine human plasmin and human plasma kallikrein the of a novel human gene product containing two Kunitz inhibitor This was designated as placental bikunin based on its sequence and the tissue of its The discovery of this protein was of the dbEST with preproaprotinin followed by of an EST to a This was used to direct the of a corresponding full-length placental cDNA that encoded the The gene encoding placental bikunin was to chromosome 19q13, it mapped adjacent to that the placental bikunin gene the of a protein was from the isolation of a functional serine protease inhibitor from human placenta which the same NH2 terminus as predicted from the placental bikunin cDNA. The cDNA for placental bikunin a protein that contains a signal a COOH-terminal to the two Kunitz domains, and a COOH-terminal hydrophilic This that the full-length protein encoded by the cDNA is to the following and may as a transmembrane In this the acid fragment be to the extracellular or of a on its NH2-terminal sequence analysis that the natural protein removal of the signal peptide predicted by the cDNA and that it been the The of the that the natural protein was in the soluble of placental using is open to the was in the natural protein but not as a transmembrane domain, or it was from the protein as a of or as has been for proteins with transmembrane such as and APP770 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, 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 of tissue Although the of the natural protein was with that predicted from the within the cloned it is of the protein sequence encoded by the cDNA is in the purified natural The presence of the NH2-terminal Kunitz was clearly by NH2-terminal and this for the protease inhibitor of the natural to the entire of the protein been by the of purified protein, we to an of the presence of the predicted domains within the native using against the NH2- and COOH-terminal Kunitz domains that the NH2-terminal is in the following the against the COOH-terminal with a at in the same that the COOH-terminal Kunitz was but not to the larger Although placental bikunin to a protein as by its from human it is that the isolation a of the an protease not purified inhibitor that was to endogenous protease to or as a of tissue The function of placental bikunin is a of at potential for in as a protein the that the protein play a role in the regulation of extracellular that are in to on the potency of the natural protein against plasma kallikrein, such the of by which the of from high weight the and the the protein function in the regulation of based on its potency against The of the biological of placental bikunin of its and protease the following members of the at the Center for acid and and