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J. Biol. Chem., Vol. 276, Issue 52, 49320-49330, December 28, 2001

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SERPINB12 Is a Novel Member of the Human ov-serpin Family That Is Widely Expressed and Inhibits Trypsin-like Serine Proteinases^*

Yuko S. Askew, Stephen C. Pak, Cliff J. Luke, David J. Askew, Sule Cataltepe, David R. Mills, Hiroshi Kato^§, Jessica Lehoczky^¶, Ken Dewar^¶, Bruce Birren^¶, and Gary A. Silverman

From the  Department of Pediatrics, Harvard Medical School, Children's Hospital, Boston, Massachusetts 02115, the ^§ Department of Obstetrics and Gynecology, Yamaguchi University School of Medicine, 1-1-1 Minami-Kogushi, Ube, Yamaguchi 755-8505, Japan, and the ^¶ Whitehead Institute for Biomedical Research/MIT Center for Genome Research, Cambridge, Massachusetts 02141

Received for publication, September 14, 2001, and in revised form, October 12, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Members of the human serpin family regulate a diverse array of serine and cysteine proteinases associated with essential biological processes such as fibrinolysis, coagulation, inflammation, cell mobility, cellular differentiation, and apoptosis. Most serpins are secreted and attain physiologic concentrations in the blood and extracellular fluids. However, a subset of the serpin superfamily, the ov-serpins, also resides intracellularly. Using high throughput genomic sequence, we identified a novel member of the human ov-serpin gene family, SERPINB12. The gene mapped to the ov-serpin cluster at 18q21 and resided between SERPINB5 (maspin) and SERPINB13 (headpin). The presence of SERPINB12 in silico was confirmed by cDNA cloning. Expression studies showed that SERPINB12 was expressed in many tissues, including brain, bone marrow, lymph node, heart, lung, liver, pancreas, testis, ovary, and intestines. Based on the presence of Arg and Ser at the reactive center of the RSL, SERPINB12 appeared to be an inhibitor of trypsin-like serine proteinases. This hypothesis was confirmed because recombinant SERPINB12 inhibited human trypsin and plasmin but not thrombin, coagulation factor Xa, or urokinase-type plasminogen activator. The second-order rate constants for the inhibitory reactions were 2.5 ± 1.6 × 10⁵ and 1.6 ± 0.2 × 10⁴ M¹ s¹, respectively. These data show that SERPINB12 encodes for a new functional member of the human ov-serpin family.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The serpin¹ superfamily contains over 500 members and has representatives in the genomes of most metazoa, plants, and certain viruses (reviewed in Ref. 1). Family members are easily identified by amino acid sequence alignments due to their high degree of structural conservation. The serpin tertiary structure consists of three -sheets, approximately nine -helices, and several loops that are arranged into a metastable conformation. Serpins employ a unique suicide-substrate-like inhibitory mechanism to neutralize their target proteinases. The mobile reactive site loop (RSL), which is perched on the surface of the molecule, serves as the pseudo-substrate and binds to the active site of the proteinase. Upon RSL cleavage, the serpin undergoes a major conformational rearrangement that traps the proteinase in a covalent acyl-enzyme intermediate (2). Serpins utilize this inhibitory mechanism to regulate proteinase cascades involved in blood clotting, fibrinolysis, complement activation, cell motility, inflammation, and cell death (1, 3, 4).

In 1993, Remold-O'Donnell (5) reported on a subset of serpins with a high degree of sequence similarity to chicken ovalbumin. Unlike the canonical serpin 1-antitrypsin (SERPINA1), members of the ov-serpin subfamily lack both classic N-terminal signal peptides and long N- and C-terminal extensions. The ov-serpins also contain a variable length loop between helices C and D that may confer functional motifs involved in, for example, nuclear localization (6) or transglutamination (7). Also, most of the ov-serpins appear to reside intracellularly with a cytoplasmic (8) or nuclear-cytoplasmic distribution (9). To date, 11 human ov-serpins (SERPINB1-10 and SERPINB13) have been cloned and sequenced. They reside within two chromosomal clusters located at 6p25 (10) and 18q21.3 (11). With the possible exception of SERPINB5 (maspin), all of the human ov-serpins inhibit various serine or cysteine proteinases and are involved in biological processes such as the inhibition of cell migration, protection against certain programmed cell death pathways and the neutralization of endogenous granule proteinases that leak into the cytosol (1, 12).

The availability of a draft DNA sequence provides an opportunity to determine whether additional ov-serpin family members exist within the human genome (13). By simple electronic hybridization using the BLAST algorithm, we identified two new human ov-serpin genes, SERPINB11 and SERPINB12, that were not detected by the available gene prediction programs. In this manuscript, we report the cloning and expression of SERPINB12. SERPINB12 encodes for a 405-amino acid protein with a molecular mass of ~46 kDa. SERPINB12 is widely expressed in many human tissues and is a potent inhibitor of trypsin-like serine proteinases.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

cDNA Isolation and DNA Sequencing-- The SERPINB12 cDNA containing the putative open reading frame was amplified as described previously (14) from testes cDNA (CLONTECH Laboratories Inc., Palo Alto, CA) using specific primers (forward 1, 5'-TATAGGATCCATGGACTCTCTTGTTACAGC-3'; reverse 1, 5'-ATATATCTCGAGTTAAGGAGAGCAGACCCTG-3'). The resulting PCR fragment was subcloned into pBluescript (Stratagene, La Jolla, CA) and analyzed using an PRISM 377 DNA sequencer (ABI, Foster City, CA) and the automated DNA sequencing facility of the Mental Retardation Research Center, Children's Hospital (Boston, MA). Sequences were analyzed using MacVector 6.0 (Accelrys Inc., Princeton, NJ), ClustalW 1.8 (15) and BLAST programs (National Center For Biotechnology Information, http://www.ncbi.nlm.nih.gov/).

Tissue Expression Survey-- cDNA prepared from 26 tissues (multiple tissue cDNA panels, CLONTECH) was assayed for SEPINB12 transcripts by nested PCR. Initially, samples were assayed using the SERPINB12-specific primers, forward 2 (5'-CAAGGAACTCTTCAGCAAGG-3') and reverse 1. For added specificity, the resulting 721-bp fragment was re-amplified using internal primers (forward 3, 5'-GAGTGTGAAGATGATGACGC-3'; reverse 2, 5'-TTTGGGTTTTGTTGTGTCTAAT-3'). The size of the nested PCR product was 527 bp. To control for integrity of the starting cDNA templates, samples were assayed for a 616-bp -actin (ACTB) fragment (forward primer, 5'-CCTCGCCTTTGCCGATCC-3'; reverse primer, 5'-GGATCTTCATGAGGTAGTCAGTC-3') as described previously (16).

Production of Recombinant SERPINB12-- SERPINB12 was re-amplified using primers (forward, 5'-TATATAGCATGCGACTCTCTTGTTACAGC-3'; reverse, 5'-TATAAAGCTTAGGAGAGCAGACCCTGCC-3') that facilitated in-frame insertion into the pQE-30 6×His tag bacterial expression vector (Qiagen Inc., Valencia, CA). The PCR fragment was digested with HindIII and SphI and ligated 3' of the 6×His tag DNA sequence. Recombinant clones were analyzed by DNA sequencing to verify that the coding sequence of SERPINB12 was intact and in-frame with the 5' 6×His tag.

The recombinant 6×His-tag-SERPINB12 fusion protein was selectively purified by metal chelate affinity chromatography using HiTrap, Ni²⁺-charged-agarose column, (Amersham Biosciences, Inc., Piscataway, NJ). Briefly, recombinant plasmid was transformed into M15 Escherichia coli and plated on LB agar containing 100 µg/ml ampicillin and 25 µg/ml kanamycin. The transformed cells were inoculated into 1 liter of LB broth containing 100 µg/ml ampicillin and 25 µg/ml kanamycin and shaken at 37 °C until A₆₀₀ = 0.5-0.7. Isopropyl-1-thio--D-galactopyranoside (1 mM final concentration) was added, and the cells were shaken another 4 h at 37 °C. Cells were collected by centrifugation (7000 × g for 10 min at 4 °C), resuspended in 10 ml of ice-cold Start buffer (1× phosphate buffer (20 mM phosphate, 0.5 m NaCl), pH 7.4, 10 mM imidazole pH 7.4) and disrupted by sonication. The cellular debris was pelleted by centrifugation (12,000 × g for 10 min at 4 °C), and the resulting supernatant was passed through a low protein binding 0.45-µm filter (Pall Gelman Laboratories, Ann Arbor, MI). The filtrate was loaded onto a HiTrap, Ni²⁺-charged-agarose column. The column was washed with 10 ml of Start buffer, and the bound protein was eluted in 5 ml of elution buffer (1× phosphate buffer, pH 7.4, 500 mM imidazole, pH 7.4). The eluate was diluted in 1× phosphate buffer to reduce the imidazole concentration to 10 mM and then passed over a second Ni²⁺-charged-agarose column. SERPINB12 was eluted using a 1× phosphate buffer-imidazole step gradient that ranged in concentration from 10 to 500 mM.

Thermostability Assays-- Aliquots of recombinant SERPINB12 were incubated for 5 min at temperatures ranging from 25 to 95 °C as described previously (17). The solution was centrifuged (12,000 × g for 10 min at 4 °C), and the supernatant was analyzed by SDS-PAGE (see below).

Enzymes, Substrates, and Buffers-- Human plasmin; human trypsin; human cathepsin (cat) G, B, K, and L; human chymotrypsin; human kallikrein; human neutrophil elastase (HNE); and subtilisin A were purchased from Athens Research & Technology, Inc. (Athens, GA). Bovine trypsin, papain, and urokinase-type plasminogen activator (uPA) were purchased from Worthington Biochemical (Lakewood, NJ), Roche Molecular Biochemicals (Indianapolis, IN), and Sigma Chemical Co. (St. Louis, MO), respectively. Thrombin and coagulation factor Xa were purchased from Calbiochem-Novabiochem (San Diego, CA). Enzyme substrates were purchased from Sigma (succinyl-Ala-Ala-Pro-Phe-para-nitroanilide (Succ-AAPF-pNA), methoxy-Succ-Ala-Ala-Pro-Val-pNA (MeO-Succ-AAPV-pNA), and d-Val-Leu-Lys-pNA (VLK-pNA)); Bachem Bioscience, Inc. (King of Prussia, PA) (H-Glu-Gly-Arg-pNA (EGR-pNA)); and Molecular Probes, Inc. (Eugene, OR) (benzyloxycarbonyl-Lys-SBenzylester, (Z-Pro-Arg)₂-R110, and (Z-Phe-Arg)₂-R110); and Enzymes Systems Products (Livermore, CA) (L-pyroglutamyl-Gly-Arg-7-amino-trifluoromethyl coumarin).

PBS (137 mM NaCl, 27 mM KCl, 10 mM phosphate buffer; pH 7.4) was used in enzymatic reactions with trypsin, plasmin, catG, HNE, chymotrypsin, kallikrein, and thrombin. Cathepsin reaction buffer (50 mM sodium acetate, pH 5.5, 4 mM dithiothreitol, 1 mM EDTA) was used with papain and catB, K, and L. Unique reaction buffers were used with uPA (100 mM Tris-HCl, 100 mM NaCl, 0.1% polyethylene glycol 6000 and 20 mM D-mannitol), subtilisin A (PBS, 0.1% Tween 20), and coagulation factor Xa (50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 0.01% Tween 20).

Determination of Protein Concentrations-- Trypsin and plasmin were calibrated using 4-methylumbelliferyl-para-guanidinobenzoate (Sigma) (18). Active site titrations were performed using a fluorescence spectrophotometer (F-3010, Hitachi Instruments, Inc., San Jose, CA) with a band pass of 10 nm and excitation and emission wavelengths of 360 and 450 nm, respectively. Assays were performed in assay buffer (50 mM Tris-HCl, 20 mM CaCl₂, 100 mM NaCl), and the activity of the proteinase was determined by calibration with a standard curve of 4-methylumbelliferone (Sigma). The concentration of recombinant SERPINB12 was determined by using the Bio-Rad Protein Assay Kit II (Bio-Rad, Hercules, CA).

Screening Assays for Enzyme Inhibition-- The screening for SERPINB12 inhibitory activity was determined initially by mixing enzyme and inhibitor in appropriate buffer, incubating for 30 min at 25 °C, and measuring residual enzyme activity as described (19). Residual enzyme activity was determined by adding the appropriate substrate and measuring its hydrolysis over time (velocity) using either a THERMOmax (in the case of -pNA and -SBzl substrates) or fmax, (in the case of -R110 and -AFC substrates) microplate reader (Molecular Devices, Sunnyvale, CA). For the UV-visible substrates, -pNA and -SBzl, wavelengths of 405 and 340 nm were used, respectively. For the fluorescent substrates, -R110 and -AFC, the excitation and emission spectra were 485 nm/538 nm and 390 nm/510 nm, respectively. The concentrations of enzyme, inhibitor, and substrate are listed in Table I, and the buffers are listed above. Percent enzyme inhibition = 100 × [1  (velocity of inhibited enzyme reaction/velocity of uninhibited enzyme reaction)].

Binding Stoichiometries-- Assays for binding between SERPINB12 and either human trypsin (25 nM) or plasmin (50 nM) were performed in a volume of 100 µl in 96-well microtiter plates (Costar 9017, Costar, Cambridge, MA). The inhibitor (concentration range 0-50 nM) was incubated with enzyme for 30 min at 25 °C. The substrate was added to a final concentration of 1 mM. The velocity of substrate hydrolysis was measured using the appropriate microplate reader. The partitioning ratio of the inhibitor-enzyme association was determined by plotting the fractional activity (velocity of the inhibited enzyme reaction/velocity of the uninhibited enzyme reaction) versus the ratio of the inhibitor to enzyme ([I]₀/[E]₀) (20). The x-intercept (i.e. the stoichiometry of inhibition (SI)) was determined by linear regression analysis.

Enzyme Kinetics-- The interaction of SERPINB12 with human trypsin or plasmin was determined by the progress curve method (21). Under these pseudo-first-order conditions, a constant amount of enzyme (either 25 nM human trypsin or 50 nM plasmin) was mixed with different concentrations of inhibitor (either 0-200 nM with trypsin or 0-700 nM with plasmin) and substrate (final concentration 1 mM). The rate of product formation was measured using the microplate reader. Because the inhibition of human trypsin is assumed to be irreversible over the course of the reaction, product formation is described as shown below, where the amount of product formation (P) proceeds at an initial velocity (v_z) and is inhibited over time (t) at a rate (k_obs),

(Eq. 1)

For each combination of enzyme and inhibitor, a k_obs was calculated by nonlinear regression of the data using Equation 1. A second-order rate constant (k') was determined by plotting a series of k_obs versus the respective inhibitor concentration and measuring the slope of the line (k' = k_obs/[I]). Because the inhibitor is in competition with substrate and the rate depends on the SI, the second-order rate constant (k') was corrected for the substrate concentration, SI, of the enzyme and inhibitor and the K_m of the enzyme for the substrate to calculate the k_a as follows,

(Eq. 2)

The K_m of human trypsin for EGR-pNA in PBS was 500 µM and that of plasmin for VLK-pNA in PBS was 1 mM . All kinetic studies were repeated at least three times.

SDS-PAGE and Immunoblotting-- Proteins were mixed with 2× gel loading buffer (4% SDS, 20% glycerol, 120 mM Tris-HCl, pH 6.8, 0.01% bromphenol blue, 2% -mercaptoethanol), heated to 95 °C for 5 min, and separated by SDS-PAGE (7.5% acrylamide, %T:%C = 19:1) according to the method of Laemmli (22). The running buffer (pH 8.3) was 25 mM Tris-base, 250 mM glycine, and 0.1% SDS. Protein bands were visualized after staining in a solution containing 0.25% Coomassie Brilliant Blue R-250, 45% methanol, and 10% acetic acid. For immunodetection, proteins separated by SDS-PAGE were electroblotted at 100 V for 1 h at 4 °C onto reinforced nitrocellulose (NitroPure, Osmonics Inc., Westborough, MA) as described (23). The transfer buffer was 25 mM Tris-base, pH 8.0, and 190 mM glycine, pH 8.3. The immunodetection of SERPINB12 was performed using a monoclonal mouse anti-histidine-tag antibody (Sigma) diluted 1/2000 as the primary antibody and a horseradish peroxidase-linked anti-mouse antibody (Amersham Biosciences, Inc.) diluted 1/2500 as secondary antibody. The immunoblot was visualized using the ECL detection kit (Amersham Biosciences, Inc.).

Matrix-associated Laser Desorption Ionization Mass Spectroscopy (MALDI-MS)-- Human trypsin (0.7 µM) in PBS reaction buffer (20 µl) was mixed with SERPINB12 (7 µM) for 20 min at 25 °C and lyophilized. For plasmin, 0.5 µM of enzyme was mixed with 5 µM SERPINB12. The mixture components were separated by MALDI-MS at the Wistar Protein Microchemistry Facility (Philadelphia, PA).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Identification of a Novel Human ov-serpin-- The presence of 11 human ov-serpin genes residing at either 6p25 or 18q21 prompted us to consider whether additional ov-serpin genes were present in the human genome. The availability of a draft sequence of the human genome (13) provided the opportunity to screen vast segments of genomic DNA sequence for serpin-like motifs without a priori insight into expression patterns. Both the hinge region of the RSL and the serpin signature motif (PROSITE: PDOC00256 ([LIVMFY]-X-[LIVMFYAC]-[DNQ]-[RKHQS]-[PST]-F-[LIVMFY]-[LIVMFYC]-X-[LIVMFAH])) are highly conserved and reside in the terminal exon of all human ov-serpin genes. To identify novel ov-serpin genomic sequences, we screened the unfinished high throughput genomic sequence data base of GenBank^TM using the BLAST2 algorithm (http://www.ncbi.nlm.nih.gov/BLAST/) using the terminal exon (exon 8) of SERPINB4 (SCCA2) as the query sequence. A significant match was detected in DNA sequence from the overlapping BAC clones RP11-519E18, -851B10, and -1117D15. The sequence from these clones corresponds to GenBank^TM accession numbers AC019355, AP001404, and AC015536, respectively. Because no new serpins were identified in the annotated DNA sequence of these clones (MapViewer (http://www.ncbi.nlm.nih.gov/)), we focused our gene-finding efforts to an ~18-kb DNA segment from AC019355 that was 5' and contiguous to the putative terminal exon of the new serpin. Using a combination of gene prediction programs (MetaGene (http://ares.ifrc.mcw.edu/MetaGene/)) and simple pairwise BLAST alignments between the 18-kb segment of AC019355 and individual exons from SERPINB4, a novel serpin gene containing at least seven exons was identified (Fig. 1). This gene was assigned the symbol, SERPINB12, by the HUGO Gene Nomenclature Committee (http://gene.ucl.ac.uk/nomenclature/).

View larger version (38K): [in this window] [in a new window]   Fig. 1.   Genomic and cDNA organization of SERPINB12. The cDNA nucleotide sequence is capitalized, and the amino acid identities and positions are located above the codons. Numbering is based on the cDNA sequence and begins with the translational start site in exon 2. Intron donor and acceptor splice sites are shown in lowercase. The intron length is in parentheses. The putative P1-P1' bond is noted by a triangle. Possible N-linked glycosylation sites and a type 2 transmembrane segment are underlined and boxed, respectively.

To determine whether a transcript for SERPINB12 could be identified, we designed oligonucleotide primers encompassing the putative start and stop codons. A fragment of ~1200 bp was amplified by PCR from a human testis cDNA library. The fragment was subcloned into pBluescript and sequenced. The cDNA sequence was identical to that predicted by the genomic sequence (Fig. 1). The deduced amino acid sequence of 405 residues predicted a M_r = 46,276 and a pI = 5.36. Alignment of the amino acid sequence of SERPINB12 with those of the other B clade members confirmed that it was an ov-serpin (Fig. 2). Conserved ov-serpin features included 1) the absence of an N-terminal hydrophobic signal sequence and N- or C-terminal extensions, 2) a Ser rather then an Asn at the penultimate residue, and 3) a CD loop (Fig. 2) (5). Other notable features of SERPINB12 were six potential N-linked glycosylation sites and a possible type 2 transmembrane helix (residues 27-46) (Fig. 1). Possible N-terminal transmembrane segments for ov-serpins have been described recently (24). SERPINB12 also appeared to contain a functional RSL that was characterized by a hinge region rich in alanines, and Arg and Ser residues at the putative P1 and P1' positions, respectively.

View larger version (116K): [in this window] [in a new window]   Fig. 2.   Amino acid alignment of SERPINB12 with other human ov-serpins. Amino acid sequences were aligned using ClustalW 1.8. The SeqVu 1.01 program (J. Gardner, Garvan Institute of Medical Research, Australia) was used to display the alignment. Colors indicate polar (green), nonpolar/hydrophobic (yellow), acidic (red), and basic (blue) residues. The CD loop is underlined with a thick line. The RSL is underlined and numbered from P17 to P3'. The putative scissile bond is marked by an arrow.

Based on their genomic structure, the human ov-serpins can be divided into two classes (10). One class contains eight exons with conserved splice site phasing (0, 0, 0, 0, 1, 0, and 0). The second class is similar except that the intron between exons 3 and 4 is not present. The splice site phasing is otherwise conserved (0, 0, -, 0, 1, 0, and 0). In both the eight-exon- and the seven-exon-containing ov-serpin genes, the translational start site is located within exon 2. Based on this comparison, SERPINB12 belongs to the eight-exon class of ov-serpin genes (Fig. 3).

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Intron-exon boundaries of human ov-serpins. The maps of the 8-exon (top) and 7-exon (bottom) containing ov-serpin cDNAs are compared with that of SERPINB12 (middle). The thick horizontal bars represent coding sequence, and the thin lines represent 5'- and 3'-untranslated sequence. The translational start sites are noted by an inverted triangle. The vertical lines indicate the positions of splice sites, with intron phasing noted above these sites. Exons are numbered below the lines. Note that the exon numbering scheme was retained in all maps to facilitate comparisons between the classes with (top) or without (bottom) an intron between exons 3 and 4. The actual exon numbers for the seven-exon-containing ov-serpins are in parentheses. Based on this comparison, SERPIN12 belongs to the eight-exon ov-serpin class.

The ov-serpin Cluster at 18q21.3-- The SERBINB12-containing BAC clones described above mapped to 18q21.3. This was expected, because all known ov-serpin genes mapped to either 6p25 or 18q21.3. Using MapViewer, we determined that SERPINB12 was 75 kb telomeric to SERPINB5 (maspin) and 35 kb centromeric to SERPINB13 (headpin) (Fig. 4). Overall, there are now 10 ov-serpin genes (including the unpublished sequence of SERPINB11) within a 775-kb segment of 18q21.3 (Fig. 4). With the exception of the local orientation of SERPINB3 (SCCA1) and SERPINB4 (SCCA2), the order of ov-serpin genes in MapViewer is in agreement with that described by previous physical mapping studies (11, 25). In the mapping studies, SERPINB3 was found to be just telomeric to SERPINB4 by virtue of the former genes loss in a congenital deletion syndrome (Fig. 4) (26). However, this order is reversed in the current version of MapViewer. Completion of the genomic sequence should help resolve this small discrepancy.

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Genomic map of the 18q21.3 ov-serpin region. The positions of the 10 ov-serpin genes located at 18q21.3 are indicated on the kilobase-scale map. The arrowheads indicate transcriptional orientation.

Tissue Expression Pattern of SERPINB12-- To determine the expression patterns of SERPINB12, hemi-nested PCR assays were performed on first-strand cDNA from 26 human tissues. The appropriately sized 528-bp PCR fragment was detected in many adult and fetal tissues, including brain, various lymphoid tissues, heart, lung, liver, pancreas, kidneys, ovary, testis, and intestines (Fig. 5). For comparison, we assessed the expression pattern of SERPINB12 relative to those of the other ov-serpin family members (Table I). The expression profiles of the other ov-serpin genes were compiled by a literature survey and by analysis of the dbEST and SAGE data bases (http://www.ncbi.nlm.nih.gov/). From this analysis, it appeared that many of the ov-serpins shared overlapping expression patterns. For example, at least seven different ov-serpin genes were expressed in brain, lung, breast, pancreas, prostate, bone marrow, lymph node, colon, uterus, ovary, skin, and placenta. However, no single tissue expressed all of the ov-serpin genes.

View larger version (24K): [in this window] [in a new window]   Fig. 5.   Tissue expression of SERPINB12. PCR was performed using cDNA templates from 26 human tissues with SERPINB12 (527 bp) and ACTB (616 bp) primers. Tissue sources are labeled above each lane of the ethidium bromide-stained agarose gels. Controls for SERPINB12 included no cDNA and SERPINB12 cDNA (+). Controls for the ACTB included no cDNA and human tumor cDNA.

A Survey of SERPINB12 Inhibitory Activity-- To determine whether SERPINB12 encoded for a functional serine proteinase inhibitor, we prepared a 6×His-tag-SERPINB12 fusion protein in E. coli. In their active conformation, serpins are metastable and are denatured by heating to ~60-70 °C (1). In contrast, RSL cleaved or latent serpins are stable when incubated at a temperature  100 °C. Prior to conducting any functional studies, we obtained a thermal denaturation profile for the recombinant serpin. 6×His-tag-SERPINB12 began to precipitate from solution at 65 °C and precipitated completely when incubated at 70 °C (Fig. 6). This result suggested that the recombinant protein was in the active conformation and could be used to screen for inhibitory activity.

View larger version (42K): [in this window] [in a new window]   Fig. 6.   SERPINB12 thermal denaturation curve. Aliquots (5 µg) of purified, recombinant 6×His-tag-SERPINB12 fusion protein were incubated for 5 min at the temperatures indicated above the lanes. Each sample was centrifuged for 10 min, and the supernatants were analyzed by SDS-PAGE. The gel was stained with Coomassie Brilliant Blue R-250.

The presence of Arg-Ser at the putative reactive center (P1-P1') suggests that SERPINB12 will inhibit trypsin-like proteinases. However, it is not always possible to predict proteinase targets based solely on the residues located at the canonical P1 position. Several serpins employ more than one residue within the RSL to bind to the enzymes S1 subsite (27). Thus, we screened for 6×His-tag-SERPINB12 inhibitory activity by incubating the serpin with a panel of serine and cysteine proteinases prior to addition of the appropriate enzyme substrate (Table II). 6×His-tag-SERPINB12 inhibited completely the enzymatic activity of the trypsin-like serine proteinases; human trypsin, bovine trypsin, and plasmin; but not thrombin or coagulation factor Xa (in the presence or absence of heparin). 6×His-tag-SERPINB12 also showed modest inhibitory activity against uPA and catG. However, subsequent SDS-PAGE analysis showed that this inhibition was due to a competition reaction, with the serpin serving as a simple substrate (not shown). Unlike SERPINB3, 6×His-tag-SERPINB12 showed no inhibitory activity against the papain-like cysteine proteinases, catK or catL.

SERPINB12 Is a Classic Inhibitory Serpin-- Several features characterize the interaction of serpins with their target proteinase. The interaction occurs 1) at near 1:1 stoichiometry, 2) with a second-order rate constant  10⁴ M¹ s¹, and 3) via RSL cleavage and formation of a stable, covalent-acyl enzyme complex.

Although the interaction between an inhibitory-type serpin and its target proteinase usually results in the formation of a covalent complex (inhibitory pathway at a rate = k_i), parallel substrate reactions can occur (substrate pathway at a rate=k_s) (28). The extent to which the serpin-proteinase complexes partition down these pathways is reflected in the stoichiometry of inhibition (SI), where the SI = (k_s + k_i)/k_i. If the substrate pathway predominates over the inhibitory pathway, the SI will exceed 1, whereas in the absence of the substrate reaction the SI = 1 (29). The SI for a serpin-proteinase reaction is determined by titration of the proteinase with the inhibitor and extrapolating to the [I]₀/[E]₀ ratio that results in a complete loss of enzyme activity. When various amounts of 6×His-tag-SERPINB12 were incubated with either trypsin (human trypsin was used in all subsequent studies, but similar results were obtained with bovine trypsin (data not shown)) or plasmin the SIs were 2 and ~2.5, respectively (Fig. 7A).

View larger version (14K): [in this window] [in a new window]   Fig. 7.   Kinetic analysis of the interaction between SERPINB12 and target proteinases. A, stoichiometry of inhibition. 10 nM trypsin () was incubated with different concentrations of 6×His-tag-SERPINB12 (0-20 nM) at 25 °C for 20 min in PBS. 50 nM plasmin () was incubated with different concentrations of 6×His-tag-SERPINB12 (0-100 nM) at 25 °C for 20 min in PBS. Residual trypsin activity was measured by adding substrate (EGR-pNA) and measuring A405. Residual plasmin activity was measured by adding substrate (VLK-pNA) and measuring A405. Fractional activity was the ratio of the velocity of inhibited enzyme (vi) to the velocity of uninhibited control (vo). The stoichiometry of inhibition was determined by using linear regression to extrapolate the inhibitor and enzyme ratio resulting in complete inhibition of the enzyme. B, the interaction of trypsin with 6×His-tag-SERPINB12 was measured under pseudo-first-order conditions using the progress curve method. Human trypsin (10 nM) and the substrate EGR-pNA (1 mM ) were added to SERPINB12 at 0 nM (), 50 nM (), 75 nM (), 100 nM (), and 150 nM (). The progress of the inactivation of the enzyme at each concentration of serpin was followed by measuring the A405 of the reaction every 11 s (inset). Assuming an irreversible reaction, the first-order rate constants (kobs) were calculated by a nonlinear regression fit of each curve using Equation 2. The kobs were plotted against the inhibitor concentration, and the slope of this line was used to determine the second-order rate constant (k). By accounting for the Km of the enzyme for the substrate, a corrected second-order rate constant (ka) was calculated (Equation 2). The ka for the 6×His-tag-SERPINB12-trypsin interaction in this representative experiment was 4.2 × 105 M1 s1. The mean value of at least three experiments is reported under "Results." C, the interaction of plasmin with 6×His-tag-SERPINB12 was measured under pseudo-first-order conditions using the progress curve method. Plasmin (50 nM) and the substrate VLK-pNA (1 mM ) were added to SERPINB12 at 0 nM (), 200 nM (), 300 nM (), 400 nM (), 500 nM (), 600 nM (), and 700 nM (). The ka was determined as described above. The ka for the 6×His-tag-SERPINB12-plasmin interaction in this representative experiment was 1.8 × 104 M1 s1. The mean value of at least three experiments is reported under "Results."

To determine the rate of complex formation between SERPINB12 and either trypsin or plasmin, we performed a kinetic analysis under first-order conditions using the progress curve method (21). Trypsin or plasmin was incubated with an excess of 6×His-tag-SERPINB12 in the presence of substrate. The progress of enzyme inactivation was followed and represented as a simple decay with a rate, k_obs (Equation 1). Rate constants (k_obs) obtained at different concentrations were plotted against the inhibitor concentrations, and the slope of this line (k') and the K_m of the substrate were used to calculate the overall second-order rate constant (k_a) as described by Equation 2. The k_a values for the interaction between 6×His-tag-SERPINB12 and trypsin and plasmin were 2.5 ± 1.6 × 10⁵ and 1.6 ± 0.2 × 10⁴ M¹ s¹, respectively (Fig. 7, B and C).

Most serpins form covalent complexes with their target proteinases. These complexes are resistant to denaturation in SDS, reducing agents, and heat (30). To determine whether 6×His-tag-SERPINB12 (M_r = 47,966) forms a covalent complex with either trypsin (M_r = 22,000) or plasmin (M_r = 76,500), the serpin was mixed with each of these proteinases and then incubated at 95 °C for 5 min in the presence of 2% SDS and 1% -mercaptoethanol. Samples were analyzed by SDS-PAGE and immunoblotting with an anti-His-tag monoclonal antibody (Fig. 8). A band at M_r  70,000 was detected in the expected position for a 6×His-tag-SERPINB12-trypsin complex (Fig. 8A). A band representing the 6×His-tag-SERPINB12-plasmin complex appeared at a combined M_r  80,000-90,000 (Fig. 8B). The molecular weight of this band is less than that predicted (M_r  114,000). Most likely, this smaller band represents a cleavage product derived from the original complex, because some serpin-proteinase complexes show increased susceptibility to cleavage by residual free enzyme (31-33).

View larger version (34K): [in this window] [in a new window]   Fig. 8.   SERPINB12-proteinase complexes. Enzyme, serpin, or serpin plus enzyme were incubated at 25 °C for 5 min. Gel loading buffer (2×) was added, each sample was heated to 95 °C for 5 min, and the proteins were separated by SDS-PAGE. Proteins were transferred to nitrocellulose and immunoblotted using a mouse monoclonal antibody specific for the histidine-tag. A, trypsin alone (0.5 µg, lane 1), purified 6×His-tag-SERPINB12 fusion protein alone (2.5 µg, lane 2), and mixtures (lane 3) of 6×His-tag-SERPINB12 (2.5 µg) plus trypsin (0.5 µg). B, plasmin alone (0.5 µg, lane 1), purified 6×His-tag-SERPINB12 fusion protein alone (2 µg, lane 2), and mixtures (lane 3) of 6×His-tag-SERPINB12 (2 µg) plus plasmin (0.5 µg). Cleaved 6×His-tag-SERPINB12 (arrow) and 6×His-tag-SERPINB12-proteinase complexes (arrowhead) are indicated. The molecular weights of 6×His-tag-SERPINB12 fusion protein, trypsin, and plasmin are ~47,900, 22,000, and 76,500, respectively.

The Reactive Center of SERPINB12-- Due to the nature of the serpin inhibitory mechanism, the location of RSL cleavage site (P1-P1') usually occurs ~17 residues downstream of the highly conserved Glu residue (P17 based on archetypal serpin, 1-antitrypsin (SERPINA1)) located in the proximal hinge region (34). To determine the reactive center (P1-P1') for 6×His-tag-SERPINB12, serpin and either trypsin or plasmin were incubated, and the resulting C-terminal cleavage fragments were resolved by MALDI-MS. For each proteinase and 6×His-tag-SERPINB12 mixture, a major peak was detected at ~4584 Da (Fig. 9). These data confirmed that the Arg-Ser at the putative P1-P1' is indeed the reactive center. However, smaller peaks were detected at ~4227 Da (Fig. 9). This result suggested the Arg-Ser residues located at the P3'-P4' positions served as a secondary cleavage site for plasmin and possibly trypsin.

View larger version (17K): [in this window] [in a new window]   Fig. 9.   Reactive center of SERPINB12. 6×His-tag-SERPINB12 (2.8-3 µg) was mixed with proteinase (0.5 µg of either trypsin (A) or plasmin (B)) and incubated for 15 min at 25 °C. The reaction mixture was then analyzed by MADLI-MS. C, the reactive site loop of SERINB12 showing the location of the primary (triangle) and secondary cleavage sites (arrow) based on the MALDI-MS data.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In this report, we describe the molecular cloning and biochemical characterization of a novel member of the human ov-serpin family, SERPINB12. The ov-serpins differ from the archetypal fluid phase (circulatory) serpins such as 1-antitrypsin (SERPINA1) or antithrombin III (SERPINC1) by the lack of a cleavable N-terminal signal peptide, the absence of N- and C-terminal extensions, and Ser instead of Asn at the penultimate position (5). SERPINB12 fulfills these criteria and shows a high degree of sequence similarity to the other 11 human ov-serpins deposited in GenBank^TM as well as one unpublished sequence (SERPINB11).² The presence of this gene was revealed by scanning the high throughput genomic (draft) sequence in GenBank^TM using the nucleotide sequence of exon 8 from SERPINB4. This analysis identified ~18 kb of contiguous DNA sequence containing at least seven exons of SERPINB12. cDNA cloning confirmed the presence of the gene, because its nucleotide sequence matched exactly that deposited in the genomic data base. Interestingly, several gene detection programs failed to identify this novel gene despite the presence of a translational start site harboring a good Kozak consensus sequence (TTTTACAATGG) (35), appropriate GT-AG motifs flanking all of the introns and conserved branch site motifs preceding the acceptor splice sites.

The human ov-serpins are divided into two classes depending on whether exon 3 encodes for an extra loop (CD loop) between helices C and D (10). Those ov-serpins containing a CD loop have an additional intron that interrupts exon 3. The net result is that the human ov-serpins contain either seven or eight exons. In both classes of genes, the translational start sites are located in exon 2. Because SERPINB12 contained a CD loop and an extra intron in the expected location within "exon 3," we concluded that this new gene falls into the eight-exon-containing group.

The human ov-serpins map to two chromosomal locations. SERPINB1, -B6, and -B9 map to 6p25 (36, 37), whereas SERPINB2, -B3, -B4, -B5, -B7, -B8, -B10, and -B13 map to 18q21.3 (25). The DNA sequence from the BAC clones used to identify SERPINB12 also map to the ~800-kb serpin cluster at 18q21.3. The clustering of these genes raises the possibility of whether some form of coordinate gene regulation controls the expression of the ov-serpins. However, preliminary qualitative assessment of the tissue expression patterns of SERPINB12 along with the other human ov-serpins suggests that control of their transcriptional activity may be more complex than originally appreciated. For example, SERPINB1 and -B6 expression was detected in 23 and 28 of the 31 tissues examined, respectively. In contrast, SERPINB10 and -B13 expression were confined to five or fewer tissues. The remaining ov-serpins, including SERPINB12, were detected in the range of 10 to 18 different tissues. When individual tissues were examined for ov-serpin expression, the results also were quite variable. Of the 31 tissues examined, 28 different ov-serpin expression patterns were detected. Only the eye and larynx, pancreas and placenta, and muscle and liver, showed expression patterns that were similar to the other, respectively. On average, each tissue expressed about six serpin genes (range 1-10). Although these expression profiles will change as more sensitive detection systems are employed and as larger sample sizes are assayed under different conditions, these data underscore two general observations. First, the ov-serpins are expressed in a wide variety of human tissues. Second, most tissues employ different combinations of multiple ov-serpins to stock their anti-proteinase armamentarium.

With the exception of SERPINB5 (maspin), all of the other ov-serpins characterized to date are suicide substrate-like proteinase inhibitors (Fig. 10) (1). The amino acid sequences of the RSLs of SEREPINB7 and -B13 also predict that these proteins are bona fide proteinase inhibitors, but no studies have been reported. SERPINB12 is no exception because the residues in the RSL predicted that this protein would inhibit trypsin-like serine proteinases. This prediction proved to be correct because SERPINB12 inhibited both trypsin and plasmin. Inhibition was demonstrated by the measurement of appropriate second-order rate constants, the formation of covalent complexes between the serpin and proteinase, and binding stoichiometries of ~2:1. The slight increase in the SI (i.e. SI > 1:1) for the SERPINB12-trypsin and SERPINB12-plasmin interactions may be attributable to several factors, including residues in the RSL that might slow loop insertion or cleavages in the RSL that lead to complex destabilization. In the former case, all of the human ov-serpins, except SERPINB12 contain a Glu at the P13 position. Perhaps the presence of Gln instead of a Glu at this position slows RSL mobility when SERPINB12 interacts with certain proteinases. Slowing of loop insertion would allow the enzyme to dissociate from the complex by providing additional time for the deacylation step to occur. In the latter case, The MALDI-MS data suggested that the Arg-Ser residues at the P3'-P4' positions could serve as an alternative cleavage site. However, cleavage after P3' would yield an RSL, which is too long to trap the proteinase in a distorted fashion (34). The net result of both of these possibilities is to increase the apparent rate of the substrate reaction, thereby increasing the SI.

View larger version (42K): [in this window] [in a new window]   Fig. 10.   Comparison of RSLs and inhibitory profiles between clade B (ov-serpins) and circulatory serpins. Amino acid alignments of the RSLs and the distal hinge regions (strand C1 (str C1)) of the ov-serpins (top) are compared with those of serpins detected primarily in the circulation (bottom). The RSLs are numbered based on the Schechter and Berger scheme using 1-antitrypsin (SERPINA1) as the reference (39). The putative P1-P1' residues are boxed. Except for the Glu residue of SERPINB9, the types of residues located at the P1 positions of the ov-serpins are similar to those of the circulatory serpins. However, these similarities do not correlate strictly with the types of target proteinases inhibited by the groups. The ov-serpins inhibit trypsin-like, chymotrypsin-like, elastase-like, and granzyme B-like serine proteinases as well as some types of cysteine proteinases. The inhibitory profile of circulatory serpins is skewed toward the inhibition of trypsin-like serine proteinases.

As the DNA sequencing and annotation of the human genome nears completion, the serpin repertoire becomes better defined. SERPINB12 and SERPINB11² brings the total number of ov-serpin genes to 13 and the total number of human serpins to 34 (1). Of this group, 21 have documented inhibitory activity. Although some of the ov-serpins can be secreted under certain conditions, they all appear to have a predominant cytoplasmic or nucleocytoplasmic distribution. Thus, if we compare the inhibitory profiles of the ov-serpins to those of the other clades, we can determine whether there is a qualitative difference between the types of inhibitors located intracellularly versus those located primarily in the circulation and extracellular fluids. Biochemical studies of the 10 known inhibitory ov-serpins (including SERPINB12) show that five are inhibitors of trypsin-like serine proteinases, four are inhibitors of chymotrypsin-like serine proteinases, two are inhibitors of papain-like cysteine proteinases, and one each is an inhibitor of elastase-like serine proteinases and granzyme B/caspase-like proteinases, respectively (Fig. 10). This summary underscores the notion that serpins in general can inhibit more than one type or class of proteinase and that residues located at the canonical P1 or surrounding positions are an imperfect predictor of target specificity. In contrast to the ov-serpins, studies of the 11 inhibitory-type circulatory serpins show that ten inhibit trypsin-like serine proteinases and one each inhibits chymotrypsin-like and elastase-like serine proteinases, respectively. Although these findings are influenced in part by an ascertainment bias, they are consistent with the predominant locations of both the serpins and proteinases. Thus, it appears that the inhibitory profile of the ov-serpins is directed against a broader class of proteinases, including those associated with intracellular protein processing and homeostasis, extracellular matrix remodeling, and activation of programmed cell death pathways. In addition, the ov-serpins may protect against mediators of inter- or intracellular injury by neutralizing proteinases stored in secretory granules or lysosomes (12, 38). In contrast, the inhibitory profile of the circulatory serpins is more skewed toward the regulation of trypsin-like proteinases associated with the clotting, fibrinolytic, and inflammatory cascades present in human plasma. The identification of naturally occurring mutations associated with phenotypic derangements and the manipulation of ov-serpin genes in simpler model organisms will go a long way in testing this hypothesis.

	FOOTNOTES

^* This work was supported by Grants CA87006 and CA86002 from the NCI, National Institutes of Health (NIH) and by Grants HD07466, HD28475, and HD18655 from the NICHD, NIH.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EMBL Data Bank with accession number(s) AF411191.

To whom correspondence should be addressed: Children's Hospital, 300 Longwood Ave., Enders 970, Boston, MA 02115-5737. Tel.: 617-355-6416; Fax: 617-355-7677; E-mail: gary.silverman@tch.harvard.edu.

Published, JBC Papers in Press, October 16, 2001, DOI 10.1074/jbc.M108879200

¹ The abbreviations and trivial name used are: serpin, serine proteinase inhibitor; RSL, reactive site loop; ACTB, -actin; cat, cathepsin; HNE, human neutrophil elastase; uPA, urokinase-type plasminogen activator; pNA, p-nitroanilide; Succ-AAPF-pNA, succinyl-Ala-Ala-Pro-Phe-p-nitroanilide; MeO-Succ-AAPV-pNA, methoxy-Succ-Ala-Ala-Pro-Val-pNA; VLK-pNA, d-Val-Leu-Lys-pNA; EGR-pNA, H-Glu-Gly-Arg-pNA; (Z-PR)₂-R110, (Z-Pro-Arg)₂-R110; (Z-FR)₂-R110, (Z-Phe-Arg)₂-R110; SI, the stoichiometry of inhibition; MALDI-MS, matrix-associated laser desorption ionization mass spectroscopy; DTNB, 5,5'-dithiobis(nitrobenzoic acid); PBS, phosphate-buffered saline; CD, loop between helices C and D.

² S. Cataltepe and G. A. Silverman, unpublished.

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