Serine proteinase inhibitors or serpins are a ubiquitous
superfamily of homologous proteins that resemble

-proteinase inhibitor (
-PI) (
)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
-P
` 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
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 µg of DNA
were used as template for PCR reactions. DNA samples were adjusted to
PCR buffer conditions in a total volume of 50 µl 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 
-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

-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

-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 5`- and 3`-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 3`- and 5`-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. 1are indicated in
parentheses): 1) CA GTG GGC CTT CAA CTC TAC (707-726); 2) C AGT
GCA GAC ATG ATG GAG TTG TA (831-854); 3) GAC AGT TAT GAT CTC AAG
TCA ACC (892-915); 4) AA TTC AAT GGA TGG GAC TCT
(1109-1090); 5) ATT CAG CTT CTC ATA GGT GAT GGC (822-799);
6) TAG TAT AAG CAG GCT GAG GTC ACG (759-736); 7) GG CTT TTC TGT
GGT GTT TTG CAC TAA (617-592). 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 5`- and 21 further 3`-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 5`-extension 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 5`-extension).
Figure 1:
Nucleotide and predicted
amino acid sequence of bomapin. The putative reactive site
P
residue is boxed. The positions of the primers
used in the initial PCR that led to the isolation of bomapin cDNA are
indicated by arrows above the nucleotide
sequence.
Northern Blotting and PCR Analysis of Bomapin mRNA in
Different Tissues
Poly(A)
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
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
10
cpm/µg using
[
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
SSPE, 5
Denhardt's solution, 0.5% SDS, 50
µg/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
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 µg of poly(A)
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
SSC,
0.1% SDS (50 °C) or 2
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
5`-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(+) (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 [
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, 6, 7, 8, 11) ,
one set of three sense and two sets of two antisense PCR primers were
designed (refer to Fig. 1and Fig. 2for 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 5`- and 3`-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 5`- (sense) and 3`-
(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.
Figure 2:
Alignment of the bomapin amino acid
sequence with other serpins. Pairwise alignments between

-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 
-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
residues are boxed.
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 
-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 
-PI; (ii) serine rather than
asparagine in the penultimate position; (iii) a variable residue rather
than valine at Val-388 in 
-PI; (iv) lack of an
N-terminal extension, beginning at amino acid 23 relative to

-PI. Alignment of the deduced primary structure of
bomapin with other serpins indicates that the reactive site
P
-P
` residues are Arg-362 and Ile-363,
respectively (Fig. 2). The proximal reactive loop hinge region,
located N-terminal from the P
residue between the P
and P
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 
-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. 2indicates 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.
Figure 4:
Bomapin forms SDS-stable complexes with
thrombin and trypsin. The complete bomapin coding region was expressed
by in vitro transcription/translation in the presence of
[
S]methionine. Samples (2.5 µl) of reaction
mixtures generated in the absence (lanes 1 and 2) and
in the presence (lanes 3-10) of the bomapin expression
construct were diluted with 5 µl of Tris-buffered saline and
incubated (15 min, 37 °C) without protease (lanes 1 and 3) and in the presence of thrombin (0.2 pmol, lane 4;
1 pmol, lane 5; 4 pmol, lanes 2 and 6; 20
pmol, lane 7) or trypsin (0.2 pmol, lane 8; 1 pmol, lane 9; 4 pmol, lane 10). Samples were heated to 100
°C for 3 min in the presence of 2% SDS and 100 mM dithiothreitol, proteins were resolved by SDS-polyacrylamide gel
electrophoresis (9% separating slab gel), and detected by
fluorography.
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 µg and 10 ng DNA in a 50 µl
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 possibility 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.
Figure 3:
Detection of the bomapin transcript in
bone marrow. Panel A, a Northern blot of 2 µg of
poly(A)
RNA isolated from human bone marrow was
hybridized to a random decamer-primed
P-labeled cDNA probe
corresponding to nucleotides 643-977 of the bomapin sequence and
exposed for 24 h to autoradiography film. Panel B, human bone
marrow, placenta, and brain cDNA libraries in the vector pGAD10
(Clontech) were used at the indicated DNA quantities as a template for
50-µl PCR amplifications utilizing specific primers to amplify the
coding regions of bomapin (upper panel) and CAP as a control (lower panel). 10 µl of the PCR products were subjected to
electrophoresis on a 1% agarose gel and visualized by ethidium bromide
staining.
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
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
[
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
SDS-stable 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

-antiplasmin(27) , and it is possible that
Asp-360 (P
position in the alignment in Fig. 2) acts
as the P
residue in an interaction between bomapin and an
ICE-related protease.