Molecular cloning of bomapin (protease inhibitor 10), a novel human serpin that is expressed specifically in the bone marrow.

Serine proteinase inhibitors or serpins are a superfamily of homologous proteins that are for the most part involved in the regulation of proteolytic processes in a variety of biological systems. Utilizing a polymerase chain reaction-based strategy we have cloned a novel member of the ovalbumin family of serpins from a human bone marrow cDNA library. The new gene encodes a 397-amino acid protein, designated bomapin, with a calculated molecular mass of 45 kDa and 48% amino acid identity with plasminogen activator inhibitor-2, human leukocyte elastase inhibitor, and cytoplasmic antiproteinase. A single 2.3-kilobase bomapin transcript is highly expressed in human bone marrow cells but was undetectable in all other analyzed human tissues. In vitro transcription and translation of the bomapin cDNA revealed the synthesis of an appropriately sized protein that was able to form SDS-stable complexes with thrombin and trypsin. The restricted expression of bomapin to the bone marrow raises the possibility that this serpin may play a role in the regulation of protease activities during hematopoiesis.

Serine proteinase inhibitors or serpins are a superfamily of homologous proteins that are for the most part involved in the regulation of proteolytic processes in a variety of biological systems. Utilizing a polymerase chain reaction-based strategy we have cloned a novel member of the ovalbumin family of serpins from a human bone marrow cDNA library. The new gene encodes a 397-amino acid protein, designated bomapin, with a calculated molecular mass of 45 kDa and 48% amino acid identity with plasminogen activator inhibitor-2, human leukocyte elastase inhibitor, and cytoplasmic antiproteinase. A single 2.3-kilobase bomapin transcript is highly expressed in human bone marrow cells but was undetectable in all other analyzed human tissues. In vitro transcription and translation of the bomapin cDNA revealed the synthesis of an appropriately sized protein that was able to form SDS-stable complexes with thrombin and trypsin. The restricted expression of bomapin to the bone marrow raises the possibility that this serpin may play a role in the regulation of protease activities during hematopoiesis.
Serine proteinase inhibitors or serpins are a ubiquitous superfamily of homologous proteins that resemble ␣ 1 -proteinase inhibitor (␣ 1 -PI) 1 in overall structure (1). In general, serpins contain a highly exposed reactive site loop near the C terminus of the molecule that interacts as a pseudosubstrate for the target protease. The inhibitory specificity of the serpin is mainly defined by the P 1 -P 1 Ј amino acid residues in the reactive site loop. Interaction of these amino acids with the active site of the target protease triggers a dramatic conformational change in the serpin that results in a stoichiometric 1:1 inhibitory complex with the protease, which is typically stable to treatment with denaturants such as SDS (2). In addition to the serpins that regulate protease activity, several members of this superfamily lack a protease inhibitory capability, e.g. angiotensinogen and ovalbumin (1,3). Ovalbumin represents the parent prototype of a unique family of proteins within the serpin superfamily, which have several structural features in common, including the absence of a typical cleavable signal sequence (4). Previously identified members of this ovalbumin family of serpin proteins (ov-serpins) include plasminogen activator inhibitor-2 (PAI-2) (5), human leukocyte elastase inhibitor (6), and squamous cell carcinoma antigen (7). More recently, a tumor suppressor called maspin (8), which is presumably not a protease inhibitor (9, 10), and cytoplasmic antiproteinase or CAP (11), also known as placental thrombin inhibitor (12) or as protease inhibitor 6 (13), have been identified as ov-serpins.
Although cytoplasmic proteases play a key role in a variety of cellular functions (14,15), little is known about the physiological targets of the ov-serpins that are expressed predominantly or exclusively as intracellular proteins (i.e. PAI-2, human leukocyte elastase inhibitor, and CAP). Kumar and Baglioni (16) have shown that overexpression of PAI-2 has a moderate protective effect on tumor necrosis factor-induced cytolysis in a fibrosarcoma cell line, and these authors have speculated that this serpin may neutralize a protease that is involved in mediating the cytolytic activity of tumor necrosis factor (16). Presently, the role of proteases (e.g. interleukin-1␤-converting enzyme (ICE)-like proteases) in programmed cell death or apoptosis is an emerging concept in biology (15). Current information indicates that proteases of the ICE family cleave substrate proteins specifically after aspartate residues (15,17,18), which are absent in the reactive site loops of the known human intracellular serpins including PAI-2. Based upon the observation that the cowpox virus encodes a serpin with aspartate in the P 1 position that is capable of inhibiting ICE-related proteases, i.e. the crmA gene product (19,20), and that apoptosis in tissue culture cell lines can be inhibited by the crmA gene product (21,22), we hypothesized that an endogenous cytoplasmic serpin with a similar function may be expressed during specific stages of cellular differentiation. A polymerase chain reaction (PCR)-based homology cloning study was initiated in an attempt to identify novel ov-serpins, and we decided to utilize bone marrow as a source of mRNA from hematopoietic cells in all stages of cellular differentiation. Here we report the cloning of a novel human ov-serpin, designated bomapin for bone marrow-associated serpin, that is expressed highly specifically in the bone marrow. As suggested by the nomenclature committee of the Genome Data Base collaboration, the systematic title proteinase inhibitor 10 was assigned for bomapin to facilitate data base searches.

EXPERIMENTAL PROCEDURES
Cloning of Bomapin-A human bone marrow cDNA library in the vector pGAD10 was purchased from Clontech (Palo Alto, CA). This library had been generated from pooled sternal bone marrow from 24 Caucasians (age 16 -70). The mRNA had been reverse transcribed by oligo(dT) and random priming. The library was amplified according to the manufacturer's instructions, and 2 g of DNA were used as template for PCR reactions. DNA samples were adjusted to PCR buffer conditions in a total volume of 50 l with 30 pmol of each PCR primer and 1.25 units of Taq polymerase (Perkin-Elmer). PCR was performed for 30 cycles in an automated thermocycler (Perkin-Elmer) with cycle times of 1 min at 94°C, 1 min at 55°C, and 2 min at 72°C. The primers used in the initial PCR amplifications included the following: 1) corresponding to amino acids 216 -221 (sense) in ␣ 1 -PI: AAA CCT GTG CAA ATG ATG, AAA CCT GTA CAG ATG ATG, and AAA CCT GTG CAG ATG ATG; 2) corresponding to amino acids 324 -318 (antisense) in ␣ 1 -PI: GT CTC GGA CAT TCC AGA GA and CT CCC TGA CAT GCC TGA GA; 3) corresponding to amino acids 372-367 (antisense) in ␣ 1 -PI: GAA AAG GAA GGG ATG GTC and GAA AAG AAA GGG ATG ATC. The primer pair that was used in the PCR that led to the amplification of bomapin sequence is underlined (corresponding to nucleotides 643-660 and 983-965 in Fig. 1, respectively). PCR products were cloned into the vector pCRII utilizing a T/A cloning kit (Invitrogen, San Diego, CA) and analyzed by restriction digestion and/or DNA sequencing using M13 forward and reverse primers. The pGAD10-based primers for anchored PCR to obtain the 5Ј-and 3Ј-sequence of bomapin were ATT CGA TGA TGA AGA TAC CCC ACC (sense) and TTG CGG GGT TTT TCA GTA TCT ACG (antisense), flanking the multiple cloning site in this vector. In the course of the subsequent cloning of the bomapin 3Јand 5Ј-sequences a total of three sense and four antisense primers were designed based upon less conserved regions of the bomapin nucleotide sequence to avoid amplification of related genes (positions of the nucleotides in Fig. 1  . We cloned a total of eight bomapin-specific PCR products, which were all amplified from cDNA inserts in pGAD10 that had been generated by random priming of the bone marrow mRNA. In addition to the nucleotide sequence shown in Fig. 1, 5 further 5Ј-and 21 further 3Ј-nucleotides were obtained. Subsequently, the bomapin coding region was amplified using G TAC CAT ATG GGA TCC ATG GAC TCT CTA GCA ACA TCA ATC AAC CAG (sense; including a 16-nucleotide 5Јextension with NdeI and BamHI sites) and T GCA GTC GAC CTC GAG CAG GAT TTA GGG GGA GCA TAA TCT TCC AT (antisense; including SalI and XhoI sites in the 5Ј-extension).
Northern Blotting and PCR Analysis of Bomapin mRNA in Different Tissues-Poly(A) ϩ RNA from human bone marrow (Clontech) was separated by denaturing electrophoresis in formaldehyde-containing 1% agarose gels and transferred to nylon membrane (Hybond-N, Amersham Corp.) according to standard procedures (23). The blots were hybridized to a 32 P-labeled probe corresponding to nucleotides 643-977 of the bomapin sequence. The probe was labeled by random priming to a specific activity of 2.7 ϫ 10 9 cpm/g using [ 32 P]dCTP (Amersham Corp.), the DECAprime II DNA labeling kit (Ambion, Austin, TX), and Sephadex G-50 mini-spin columns (Worthington) for probe purification. Hybridization was performed in 5 ϫ SSPE, 5 ϫ Denhardt's solution, 0.5% SDS, 50 g/ml freshly denatured salmon sperm DNA (Life Technologies, Inc.) for 15 h at 65°C, and the blot was washed to a final stringency of 1 ϫ SSPE, 0.1% SDS at 65°C. The experiment was performed twice with similar results. Human adult multiple tissue Northern blots I and II containing 2 g of poly(A) ϩ RNA/lane (Clontech) were hybridized according to the manufacturer's protocol to the same bomapin-specific probe, and the blots were washed in two different experiments to final stringencies of 0.1 ϫ SSC, 0.1% SDS (50°C) or 2 ϫ SSC, 0.05% SDS (50°C), respectively. Hybridization to a 2.0-kb human ␤-actin cDNA probe (Clontech), labeled as described above, was used as a positive control to confirm approximately equal mRNA loading in all lanes. Blots were exposed for up to 10 days to autoradiography film (XAR-5, Kodak). Templates for PCR amplifications were human bone marrow, placenta, and brain cDNA libraries in pGAD10 (Clontech). PCR was carried out as described above using the primers to amplify the complete coding region of bomapin. Primers for control amplifications of the CAP cDNA were based on the published sequence (11), had 12-nucleotide 5Ј-extensions, and included GTA CCG GAA TTC TCT GCC ATC ATG GAT GTT CT (sense, base position 180 -199) and TA TGA AGT CGA CCT GCC CTG TCC TCA CGG AGA (antisense, base position 1330 -1311).
In Vitro Transcription/Translation of the Bomapin cDNA-The complete bomapin coding region was subcloned into the vector pBluescript SK(ϩ) (Stratagene, La Jolla, CA) using BamHI and XhoI restriction sites. The cDNA insert in this construct was expressed by in vitro transcription and translation in the presence of [ 35 S]methionine (Amersham Corp.) for 90 min at 30°C using a coupled reticulocyte lysate system (Promega, Madison, WI) and T3 RNA polymerase (Promega). Human thrombin (T6759, 3000 units/mg of protein) and bovine trypsin (T8253, type III) were obtained from Sigma.

RESULTS AND DISCUSSION
Cloning of Bomapin-Based upon three relatively conserved regions in the nucleotide sequences of the known human ov-serpins (5-8, 11), one set of three sense and two sets of two antisense PCR primers were designed (refer to Figs. 1 and 2 for the primer positions). Primers were designed to differ in up to 6 positions from the corresponding individual nucleotide sequences of the known ov-serpins to allow amplification of related sequences. Utilizing a human bone marrow cDNA library in the plasmid vector pGAD10 as the template, PCR amplifications with the 12 different possible combinations of forward and reverse primers were carried out. Products of the expected sizes for ov-serpins were detected in 11 reactions, and 2 reactions were selected based upon an unexpectedly large amount of appropriately sized PCR products in view of the mismatches between the primer sequences and the known ov-serpin cDNAs. Restriction analysis of the cloned PCR products identified an appropriately sized insert with unique restriction sites that did not match up with the known ov-serpins, and this insert was sequenced. Data base searches indicated a novel nucleotide sequence with highly significant homology to serpin proteins. The 5Ј-and 3Ј-ends of the open reading frame were then identified by anchored PCR using primers based upon the cloned insert and the vector pGAD10. PCR primers located at the 5Ј-(sense) and 3Ј-(antisense) ends of the 1191-nucleotide open reading frame were subsequently used for cDNA amplification. Both strands of cloned PCR products from three independent amplifications were completely sequenced, confirming the sequence shown in Fig. 1.
Sequence Analysis-The isolated cDNA contains a single large open reading frame that encodes a 397-amino acid protein, denoted bomapin, with a calculated molecular mass of 45.4 kDa (Fig. 1). The first ATG codon in this sequence is at the same position as the initiation codons of all ov-serpins, and it is part of a favorable sequence (ACAATGG) for translation start (24). Furthermore, in vitro translation of the open reading frame beginning with this ATG codon leads to the synthesis of a functional protein (refer to Fig. 4). Determination of the authentic translation start site will ultimately require the purification of this protein followed by N-terminal sequencing of the native protein. The deduced amino acid sequence contains two cysteine residues (Cys-68 and Cys-395) and four potential N-linked glycosylation sites (Asn-81, Asn-201, Asn-210, and Asn-383). Data base searches demonstrated a highly significant homology of the bomapin primary structure with proteins of the serpin superfamily. Sequence alignment (Fig. 2) indicates that bomapin is a typical serpin including 44 of the conserved 51 residues that were previously designated as crucial for the serpin tertiary structure (1). A notable exception is the serine residue corresponding to Pro-54 in ␣ 1 -PI. The greatest amino acid identity (48%) was found between bomapin and PAI-2, human elastase inhibitor, and CAP, suggesting that bomapin is a new member of the ovalbumin family of serpin proteins (4). All of the structural features that distinguish ov-serpins from the larger superfamily of serpin proteins (4) are also met by bomapin (Fig. 2): (i) lack of a C-terminal extension, ending with the equivalent of Pro-391 in ␣ 1 -PI; (ii) serine rather than asparagine in the penultimate position; (iii) a variable residue rather than valine at Val-388 in ␣ 1 -PI; (iv) lack of an N-terminal extension, beginning at amino acid 23 relative to ␣ 1 -PI. Alignment of the deduced primary structure of bomapin with other serpins indicates that the reactive site P 1 -P 1 Ј residues are Arg-362 and Ile-363, respectively (Fig. 2). The proximal reactive loop hinge region, located N-terminal from the P 1 residue between the P 17 and P 8 positions, is believed to play an important role in the mechanism of protease inhibition by conveying a certain degree of mobility to the reactive site loop (25). The hinge region in the primary structure of bomapin (EQGTEAAAGS) is conserved according to the consensus sequence in inhibitory serpins (9), suggesting that this novel serpin has the potential for protease inhibition. The sequence between the amino acids corresponding to residues 86 and 89 in ␣ 1 -PI is highly divergent in different serpins, and these amino acids are believed to form a loop between helices C and D in the serpin tertiary structure (1,26). The sequence alignment in Fig. 2 indicates that this interhelical loop contains 22 residues including a cluster of charged amino acids between Lys-67 and Glu-79 in the bomapin primary structure. This domain might be important for interactions between bomapin and other molecules.
Expression of Bomapin Transcript in Human Tissues-Northern blotting demonstrated the expression of a single 2.3-kb bomapin transcript in bone marrow (Fig. 3A). To determine the distribution of bomapin expression in other tissues, Northern blots were analyzed containing mRNA from the following human tissues: heart, brain, placenta, lung, liver, skeletal muscle, kidney, pancreas, spleen, thymus, prostate, testis, ovary, small intestine, colon, and peripheral blood leukocyte. However, transcripts for bomapin could not be detected in any of these tissues (data not shown). To confirm this result, PCR was utilized in an attempt to amplify bomapin-specific cDNA from human placenta and brain cDNA libraries (Fig. 3B). Utilizing the bomapin-specific primers, appropriately sized PCR products (1232 base pairs) were detected over a wide range of bone marrow template concentrations (between 1 g and 10 ng DNA in a 50 l reaction), whereas no amplification products were detected in any of the reactions using placenta or brain cDNA. In contrast, utilizing CAP-specific primers, appropriately sized PCR products (1175 base pairs) were readily detected after amplification from all three cDNA templates, including the brain cDNA. CAP expression in the brain has been observed to be either undetectable (11) or barely detectable (12) by Northern blotting. Although we cannot rule out the possi-FIG. 2. Alignment of the bomapin amino acid sequence with other serpins. Pairwise alignments between ␣ 1 -PI, bomapin, PAI-2, human elastase inhibitor (EI), and CAP were generated using the GAP routine of the Genetics Computer Group (Madison, WI) software package (gap weight, 3.0; length weight, 0.1) and adjusted manually to reduce the number of gaps in the final alignment. Amino acids are numbered according to the sequence of mature ␣ 1 -PI (1). The regions with a relatively conserved nucleotide sequence that correspond to the three primer sets that were initially designed to amplify novel ov-serpins are indicated by arrows, and the reactive site P 1 residues are boxed.
bility that bomapin mRNA is expressed in other tissues at a very low level or that it is expressed in tissues that were not analyzed, our data suggest that this novel ov-serpin is expressed highly specifically in the human adult bone marrow, presumably in hematopoietic precursor cells.
Bomapin Forms SDS-stable Complexes with Proteases-Sequence analysis indicated that bomapin is a potentially protease-inhibitory serpin (conserved hinge region) with arginine in the P 1 position of the reactive site loop, suggesting that bomapin might inhibit trypsin-like proteases. To test this hypothesis, the complete bomapin coding region was expressed by in vitro transcription/translation. Fig. 4 (lane 3) shows that a major translation product with an apparent molecular mass of 42 kDa, close to the calculated molecular mass of 45 kDa, was detected by SDS-polyacrylamide gel electrophoresis. Several lower molecular mass bands presumably represent cleaved forms of bomapin or the initiation of translation from internal ATG codons in the nucleotide sequence, which is known to occur in in vitro translation systems. No background protein incorporation of [ 35 S]methionine in the absence of exogenous DNA was detected (Fig. 4, lane 1). After incubation of the transcription/translation reactions with thrombin (Fig. 4, lanes  4 -7) and trypsin (Fig. 4, lanes 8 -10), radioactivity-containing bands were detected with an apparent molecular mass of 74 and 65 kDa, respectively. The size of these bands corresponds to the combined molecular masses of bomapin and reduced thrombin (32 kDa) and trypsin (24 kDa), demonstrating the capability of bomapin to form SDS-stable complexes with these proteases. In the presence of higher concentrations of thrombin and especially trypsin, the bomapin complexes were progressively degraded (Fig. 4, lanes 7 and 9 -10). These data indicate proper folding of the in vitro translated bomapin and demonstrate that this novel ov-serpin has the ability to form SDSstable complexes with trypsin-like proteases. However, the physiological target proteases remain to be established. Separate reactive sites for inhibitory interactions with different proteases have been described in the serpin ␣ 2 -antiplasmin (27), and it is possible that Asp-360 (P 3 position in the alignment in Fig. 2) acts as the P 1 residue in an interaction between bomapin and an ICE-related protease.