©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Gene Structure, Chromosomal Localization, and Expression of the Murine Homologue of Human Proteinase Inhibitor 6 (PI-6) Suggests Divergence of PI-6 from the Ovalbumin Serpins (*)

Jiuru Sun , John B. Rose (1), Phillip Bird (§)

From the (1)Department of Medicine, Monash Medical School, Clive Ward Centre, Box Hill Hospital, Box Hill 3128, Australia and the Department of Anatomy and Cell Biology, University of Melbourne, Parkville 3052, Australia

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Human proteinase inhibitor 6 (PI-6) is a recently described protein belonging to the serine proteinase inhibitor (serpin) superfamily. Sequence similarity suggests that PI-6 most resembles the ovalbumin (ov) serpins which include plasminogen activator inhibitor-2, the squamous cell carcinoma antigen, monocyte/neutrophil elastase inhibitor, and maspin. Although these proteins are associated with carcinomas and inflammation, they appear to have diverse functions and little is known of their physiological roles. In this study we have characterized cDNA and genomic clones encoding mouse PI-6 in order to analyze the localization, structure, and expression of the gene. The reactive center residues (Arg-Cys) are conserved in the mouse molecule, and recombinant mouse PI-6 was shown to bind thrombin, indicating that it has similar inhibitory properties to its human counterpart. Using reverse transcriptase-polymerase chain reaction assays on RNA isolated from 15-day-old embryos and adult mice, we have shown that mouse PI-6 expression is developmentally regulated, and that, unlike human PI-6, it is absent from the placenta. The mouse homologue of the human PI-6 gene has been designated Spi3 and was mapped to chromosome 13 between the Pl1 and ctla2 genes. It spans 20 kilobases, consists of 7 exons and 6 introns, and contains a TATA motif 24 nucleotides upstream of the transcriptional start site. A 680-base pair DNA fragment containing this motif and 31 nucleotides of the 5`-untranslated region of the structural gene directed transcription of a bacterial cat gene, demonstrating the presence of a functional promoter. The PI-6 gene lacks an intron present in the ovalbumin and PAI-2 genes; otherwise it is identical in terms of the numbers, position, and phasing of the intron/exon boundaries. These results suggest that PI-6 and the ov-serpin genes have diverged and do not belong to the same subgroup.


INTRODUCTION

Serine proteinase inhibitors (serpins)()are a family of proteins that regulate the activity of serine proteinases involved in extracellular processes such as coagulation, fibrinolysis, complement fixation, and embryo implantation(1) . A group of five proteins (ovalbumin, maspin, squamous cell carcinoma antigen, plasminogen activator inhibitor 2 (PAI-2), and monocyte/neutrophil elastase inhibitor) make up a branch of the serpin family known as the ovalbumin (ov) serpins(2) . The physiological roles of these inhibitors are not understood, but it is thought that they function in extracellular processes because alterations in their production or release are associated with tumorigenesis and inflammation. For example, loss of maspin correlates with increased malignancy and metastasis of breast carcinomas(3) ; increases in the levels and release of squamous cell carcinoma antigen occur in squamous cell carcinomas(4) ; and increased expression of PAI-2 occurs in monocytes during inflammation(5) . The ov serpins are unusual because each lacks an amino-terminal signal peptide normally required to initiate secretion and glycosylation of the protein. Nevertheless, secretion and glycosylation of most of the ov-serpins (ovalbumin, maspin, squamous cell carcinoma antigen, PAI-2) does occur, mediated by nonconventional signal sequences present elsewhere in the protein.

Recently, we and others have identified a new serpin that closely resembles ov-serpins in amino acid sequence and which also lacks a coventional signal peptide(6, 7, 8) . This serpin was originally designated the placental thrombin inhibitor (7) and cytoplasmic antiproteinase(8) , but to avoid confusion the Genome Data Base organization has recommended that it be known as proteinase inhibitor 6 (PI-6). Several facts suggest that, unlike most of the ov-serpins, PI-6 is an intracellular protein: it is not glycosylated (although it possesses three potential sites for asparagine-linked carbohydrate); it has never been detected in the medium of cultured cells; and it is sensitive to oxidation(7) .

It is thought that closely related serpins share common gene structures clustered at common loci and that analysis of serpin gene configuration and position is a better guide to evolutionary relatedness than amino acid similarity or physiological function(9) . For example, a group of serpins consisting of the protein C inhibitor, corticosteroid binding globulin, -1 proteinase inhibitor, and -antichymotrypsin have similar gene structures which are clustered on human chromosome 14(10) . In the case of the ov-serpins, it has been shown that chicken ovalbumin and human PAI-2, although separated by millions of years of evolution, have identical gene organizations in terms of the numbers, positions, and phasing of the intron/exon boundaries(11) . It can be predicted that PI-6 will exhibit a similar gene organization if it is a member of the ov-serpin group.

In this study we have isolated and characterized a cDNA for mouse PI-6, established the structure of the mouse gene, localized it to chromosome 13, and examined its expression in the embryo and adult. We find that it is expressed in most adult tissues and appears to be developmentally regulated. It is almost identical in gene organization with PAI-2 and ovalbumin, except that it lacks an intron present in both genes and it does not co-localize with PAI-2. This suggests that PI-6 does not belong to the ov-serpin branch of the serpin family.


EXPERIMENTAL PROCEDURES

General Methods

General recombinant DNA methods were as described in Ref. 12. cDNA probes were labeled with [-P]dCTP (DuPont NEN) using the random hexamer method (NEBlot Kit, New England Biolabs). Oligonucleotides were labeled with [-P]ATP (DuPont NEN) as described(12) . Conventional DNA sequencing was carried out using the Sequenase II system (Amersham). Cycle sequencing was carried out using the CircumVent Kit (New England Biolabs). Oligonucleotide primers for PCR and sequencing were synthesized by Bresatec (Australia).

Isolation and Sequencing of Partial Mouse PI-6 cDNAs

4 10 plaques of a mouse liver cDNA library in gt10 (Stratagene) were screened with a full-length cDNA encoding human PI-6 (7). Filters were hybridized in 50% (v/v) formamide, 6 SSC at 32 °C overnight, and washed in 2 SSC at 45 °C. Inserts from 3 positive clones were released by EcoRI digestion and subcloned into Bluescript II KS (Stratagene) for sequence analysis. All contained short (up to 600 bp) sequences homologous to the 3` end of human PI-6 cDNA. The longest fragment was then used to screen a similar number of plaques from a second mouse liver cDNA library in gt11. 10 positive clones were obtained, all containing approximately 1.2-kb inserts. Restriction mapping showed that each insert contained 2 internal EcoRI sites. Each of the three fragments was subcloned and sequenced. In addition, primers specific for gt11 were used to amplify and subclone a complete copy of the insert. Sequence analysis of each of the EcoRI fragments and the PCR fragment confirmed that a mouse homologue of PI-6 had been isolated. However, all of these clones lacked 5` sequences present in human PI-6.

To isolate the 5` end of the mouse PI-6 cDNA, anchored PCR (13) was employed. Briefly, mRNA prepared from BALB/c 3T3 cells was used as a template for the synthesis of cDNA(14) . A 50-µl reverse transcription reaction was carried out at 42 °C for 2 h and contained 1 µg of mRNA, 0.5 µg of a purified oligonucleotide (PB-92; ), 400 µM dNTP, 10 units of RNasin (Promega), and 20 units of avian myeloblastosis virus reverse transcriptase (Promega). The reaction was ethanol-precipitated and resuspended in 50 µl of HO. A portion of the cDNA was then ligated to the anchoring oligonucleotide (PB-40; ) that had been 5`-phosphorylated and blocked at the 3` end by incubation with ddATP and terminal deoxytransferase as described(13) . The 20-µl ligation reaction contained 2 µl of the cDNA, 20 pmol of the anchoring oligonucleotide, 25% (w/v) PEG 8000, and 20 units of T4 RNA ligase (New England Biolabs) and was incubated overnight at room temperature. The following day, 64 µl of 0.5 M NaCl, 10 mM Tris, pH 8, 1 mM EDTA was added to terminate the reaction. 2 µl of the terminated ligation was used as a template for PCR. This PCR (performed in duplicate) used 40 pmol of the primers PB-103 and PB-91 () and contained 50 mM Tris, pH 9.0, 50 mM KCl, 0.1% (v/v) Triton X-100, 1.5 mM MgCl, 200 µM dNTPs, and 1 unit of TthI. 35 cycles of 95 °C for 90 s, 50 °C for 60 s, and 70 °C for 90 s were performed. The resulting 400-bp products were subcloned into the plasmid pCRII (Invitrogen). Sequence analysis confirmed the presence of sequences homologous to the 5` end of human PI-6.

Construction of a Mouse PI-6 Expression Vector

A PCR-based strategy was employed to obtain a full-length mouse PI-6 cDNA. mRNA from BALB/c 3T3 cells was reverse-transcribed as above except that 0.5 µg of poly(dT) primer was used. A PCR containing 0.5 µl of the cDNA, 40 pmol of the primers PB101 and PB105 (), 20 mM Tris-HCl, pH 8.8, 10 mM KCl, 10 mM (NH)SO, 2 mM MgSO, 0.1% (v/v) Triton X-100, 200 µM dNTPs, and 2 units of Vent polymerase (New England Biolabs) was performed. The reaction was cycled 35 times at 95 °C for 90 s, 50 °C for 60 s, and 72 °C for 90 s. The resulting 1.2-kb product was cloned into the pCRII vector and sequenced completely. The cDNA was released from the pCRII plasmid by digestion with EcoRV and SpeI and was cloned into pSVTf (15) digested with SmaI and XbaI. Mouse PI-6 mRNA was synthesized in vitro from the resulting plasmid by T7 RNA polymerase and translated in a rabbit reticulocyte lysate system as described(16) .

Analysis of Mouse PI-6 RNA Distribution by Reverse Transcriptase-PCR

An adult mouse was dissected, and total RNA was isolated from various organs as described(14) . RNA was also isolated from the pooled organs of five 15-day-old embryos. Reverse transcription using 10 µg of RNA was performed as described above. To control for DNA contamination in the subsequent PCR, a parallel reaction without avian myeloblastosis virus reverse transcriptase was performed. A PCR using TthI polymerase was performed as described above except that it contained 4 primers: 5 pmol of the glyceraldehyde-3-phosphate dehydrogenase specific primers (PB-120 and 121; ), as well as 25 pmol of the mouse PI-6 primers PB101 and 105 (). 35 cycles were performed; 95 °C for 90 s, 50 °C for 60 s, and 70 °C for 90 s. The PCR products were separated on 2% agarose gels, visualized by ethidium bromide staining, and then transferred to nylon for Southern hybridization using a P-labeled oligonucleotide (PB92).

Isolation and Characterization of the Mouse PI-6 Gene

1 10 plaques from a 129/Sv mouse genomic DNA library in FIX II (Stratagene) were screened using the full-length mouse PI-6 cDNA as a probe. Conditions were as described above except that the hybridization was performed at 42 °C and the final wash was in 0.5 SSC at 65 °C. DNA was prepared from the positives using the Wizard Lambda Preps DNA Purification System (Promega). Intron/exon boundaries were identified by sequencing the two clones directly using specific oligonucleotide primers (). DNA from each clone was used as template in a cycle sequencing reaction (CircumVent; New England Biolabs). The size of each intron was estimated by analysis of the size of the products resulting from a PCR using flanking primers () in a TthI-based protocol (see above) .

Mouse PI-6 Promoter Analysis

The genomic clone 7 was digested with HindIII and a 6-kb fragment containing the 5`-flanking region, exon I, intron A, exon II, and part of intron B was subcloned into pUC118 (pUC118/7H3). A 700-bp HindIII-BglII fragment containing the 5`-flanking region, exon I, and a short region of intron A was then subcloned into the HindIII/BamHI sites of Bluescript II KS (Stratagene). The sequence of this fragment was determined on both strands using vector- and insert-specific oligonucleotide primers. A plasmid containing the 5`-flanking region of the PI-6 gene (-646 to +31) fused to the chloramphenicol acetyltransferase (CAT) gene was constructed by digestion of pUC118/7H3 with HindIII (filled) and BspE1 (filled) and ligation to Bluescript II KS digested with SmaI. The resulting plasmid was digested with ClaI and SpeI, and the insert ligated to a ClaI/XbaI fragment consisting of the vector and cat gene portion of pSVTf-CAT(17) .

BALB/c 3T3 cells were incubated in 5% CO using Dulbecco's modified Eagle's medium supplemented with 10% (v/v) fetal bovine serum and 20 mM glutamine (Life Technologies, Inc.). Cells were transfected with plasmid DNA using the calcium phosphate procedure(12) . Clones were obtained by co-transfection with a marker plasmid and selection in G418 as described(17) . Preparation of cell extracts, CAT assays, the positive control plasmid pSVTf-CAT, and the negative control plasmid p2CAT has been described previously(17) .

Localization of the Mouse PI-6 Gene

The position of the mouse PI-6 gene was mapped by analysis of The Jackson Laboratory (C57BL/B6JEi SPRET/Ei)F1 SPRET/Ei backcross DNA panel (BSS) as developed and described by Rowe et al.(18) . A PvuII restriction fragment length polymorphism between Mus musculus (C57BL/B6JEi) and Mus spretus (SPRET/Ei) was identified by Southern analysis using an XbaI-HindIII genomic fragment probe containing part of intron A, exon II, and most of intron B. Approximately 2 µg of genomic DNA from each of the 94 animals in the BSS panel was then digested with PvuII, separated by agarose gel electrophoresis, transferred to a charged nylon membrane (GeneScreen Plus, DuPont NEN), and hybridized to the same probe in 6 SSC at 68 °C. The final wash was in 1 SSC at 68 °C. The presence or absence of the M. musculus fragment in each of the animals was scored, and the results were analyzed by L. Rowe at The Jackson Laboratory using the MapManager program(19) .


RESULTS

Isolation of a cDNA for Mouse PI-6

We have previously characterized a cDNA encoding human PI-6(7) . This cDNA was used as a probe in low stringency screens of two mouse liver cDNA libraries. Although several positive and related clones were isolated from these libraries, restriction mapping and DNA sequencing showed that all were incomplete. Comparison of the sequence of the longest clone to human PI-6 showed 76% similarity at both the nucleotide and protein level. In particular it showed that the reactive center residues (P-P`; Arg-Cys) are conserved in the mouse molecule, suggesting that it inhibits the same serine proteinases (e.g. thrombin) as human PI-6. However, in comparison with the human clone, this mouse clone lacked a 5`-untranslated region and the first 11 codons.

To isolate the 5` end of a mouse PI-6 cDNA, an anchored PCR strategy was used(13) . mRNA prepared from BALB/c 3T3 cells was used as a template for cDNA synthesis primed by oligo(dT). An anchoring oligonucleotide (blocked at the 3` end by addition of ddATP using terminal deoxytransferase) was ligated to the cDNA using T4 RNA ligase, and the cDNA was then used as a template for PCR using one primer complementary to the anchoring oligonucleotide, and the other primer antisense to a 5` region of the mouse PI-6 coding sequence. A resulting 330-bp product was verified by hybridization to a specific oligonucleotide designed to a sequence upstream of the antisense (PI-6-specific) primer. DNA sequence analysis confirmed that this 330-bp fragment contained the mouse PI-6 5`-untranslated region.

The complete sequence of the mouse PI-6 cDNA was assembled from the longest partial cDNA clone arising from the libraries and from the 5` PCR product (see Fig. 1). Sequence data were derived by walking down both strands using specific oligonucleotide primers (). A 1.2-kb full-length cDNA clone was then amplified from the BALB/c 3T3 cDNA by PCR using a proofreading polymerase and primers corresponding to the 5` and 3` ends of the predicted mouse PI-6 mRNA. The resulting product was cloned and entirely sequenced. There were no differences between the 5` sequences of this full-length cDNA and the 330-bp fragment, and both matched the corresponding genomic sequence, suggesting that neither clone contained PCR-introduced mutations.


Figure 1: Sequence of the mouse PI-6 (Spi3) gene. Shown is the composite sequence of the 5`-flanking region and structural gene deduced by analysis of mouse cDNAs and two genomic clones. Intervening sequences are boxed and shown in lowercase; intron lengths were estimated by PCR. The transcriptional start site (+1) was deduced by anchored PCR as described. The TATA box (-24) and polyadenylation signals (+1292) are underlined. The vertical arrow indicates the putative reactive center bond (P-P`), and the asterisk indicates a potential site for N-linked glycosylation.



Expression of the Mouse PI-6 cDNA

To show that it could direct the synthesis of a protein, the mouse PI-6 cDNA was subcloned into the mammalian expression vector pSVTf(15) . This vector allows the expression of cDNAs either in mammalian cells under the control of the SV40 early promoter or in rabbit reticulocyte lysates following in vitro transcription using T7 RNA polymerase. In this case, PI-6 mRNA produced in vitro was used to program a rabbit reticulocyte lysate containing [S]methionine. Products from the translation were analyzed by SDS-polyacrylamide gel electrophoresis and fluorography (Fig. 2). As predicted from the sequence data, a 42-kDa protein was produced in lysates containing mouse PI-6 mRNA (lanes 3 and 4), but was not evident in unprogrammed lysates (lanes 1 and 2).


Figure 2: Expression of the mouse PI-6 cDNA. A full-length cDNA was cloned into the expression vector pSVTf (15) and linearized at the 3` end of the insert. RNA synthesized in vitro using T7 RNA polymerase was used to program rabbit reticulocyte lysates containing [S]methionine. 10 ng of thrombin were added to the indicated samples and incubated at 37 °C for 30 min. Products were separated by reducing 10% SDS-PAGE and visualized by fluorography.



To show that the protein could function as a proteinase inhibitor, thrombin was added to the lysates prior to SDS-polyacrylamide gel electrophoresis (Fig. 2, lanes 2 and 4). This resulted in the appearance of a 67-kDa species in the programmed lysates. This corresponds to an SDS-stable complex formed between thrombin and the 42-kDa protein and is reminiscent of those commonly observed between serpins and their target proteinases(1) . It also resembles the complex seen between human PI-6 and thrombin in the reticulocyte system (7) and confirms that mouse PI-6 has inhibitory properties similar to its human counterpart.

Isolation and Characterization of the Mouse PI-6 Gene

Seven overlapping clones containing fragments of the mouse PI-6 gene were isolated from a 129/Sv genomic DNA library screened at high stringency with the full-length mouse cDNA. These clones were analyzed by EcoRI and HindIII digestion and hybridization to a panel of mouse PI-6-specific oligonucleotides (PB89, -90, -91, -96, -97, -98, -99, -104, and -105; ). The entire gene spanning about 20 kb was represented in two overlapping clones (Fig. 3). Intron/exon boundaries were deduced by direct (cycle) sequencing of the clones using the oligonucleotide primers shown in and subsequent comparison of the genomic sequences to the cDNA sequence. This analysis showed that the gene consists of 7 exons and 6 introns ( Fig. 1and Fig. 3). Clone 5 contains the exons III-VII and the 3`-flanking region, while clone 7 contains the 5`-flanking region and exons I-V.


Figure 3: Map of the mouse PI-6 gene. The structure of the mouse PI-6 gene was deduced by restriction endonuclease mapping, hybridization with specific oligonucleotide probes (Table I), and DNA sequencing of two overlapping genomic clones (5 and 7). Exons are designated by Roman numerals and indicated by filled boxes. Introns are designated by uppercase letters, and the TATA box is shown as a closed circle. The RFLP probe is the segment of genomic DNA used for the gene localization studies. E, EcoRI; H, HindIII.



The length of each intron was estimated by PCR on genomic clone templates using primers corresponding to sequences in the flanking exons. The sizes of the resulting products were measured by comparison to DNA standards in agarose gel electrophoresis (data not shown). Analysis of the position and phasing of each intron/exon boundary showed that the PI-6 gene is identical in every respect with the human PAI-2 and chicken ovalbumin genes (11) with one exception: the PI-6 gene lacks an intervening sequence corresponding to the third intron present in both these genes (see Fig. 8).


Figure 8: Intron/exon structure of members of the serpin gene family. This figure is derived from that shown in Ref. 11. Thick lines indicate coding sequences; thin lines indicate untranslated regions; arrows marked by uppercase letters indicate positions of introns. Sequences are aligned on the basis of amino acid similarity with the scale showing numbers of amino acids. AT-III, antithrombin III; 1-AT, -antitrypsin; PAI-1, plasminogen activator inhibitor 1; OVAL., ovalbumin; PAI-2, plasminogen activator inhibitor 2; SPI3/PI6, proteinase inhibitor 6.



Localization and Designation of the Mouse PI-6 Gene

Recently, Rowe et al.(18) have described a panel of DNAs derived from interspecific backcross mice that can be used for mapping genetic loci in the mouse. Maps have been generated using these DNAs and anchored to other published maps. To place an unknown gene on the map, a specific PCR or restriction fragment length polymorphism between the parental species of mice (M. musculus C57BL/6JEi and M. spretus SPRET/Ei) must be found. The distribution of the polymorphism in DNA from 94 (M. musculus M. spretus)F1 M. spretus (BSS) or 94 (M. musculus M. spretus)F1 M. musculus (BSB) backcrossed mice is then analyzed to deduce the map position.

A PvuII restriction fragment length polymorphism between M. musculus and M. spretus genomic DNA was detected by hybridization of a XbaI-HindIII probe consisting of part of intron A, exon II, and part of intron B of the mouse PI-6 gene (see Fig. 3). Hybridization of this probe to a panel of 94 PvuII-digested DNAs made from backcross mice (BSS(18) ) localized the PI-6 gene to chromosome 13 (Fig. 4). Data in show the recombination frequencies for the nearest linkage markers for the mouse PI-6 gene. No recombinants were found between the PI-6 gene and the motif-primed PCR marker D13Bir8, which is positioned approximately 20 centimorgans from the centromere on chromosome 13(18) . On the map of chromosome 13, the PI-6 gene falls between the placental lactogen 1 gene (3.2 centimorgans proximal) and ctla2 gene (39 centimorgans distal).


Figure 4: Chromosome linkage map showing the location of the mouse PI-6 gene. The midregion of mouse chromosome 13 is shown as a solid line. Markers in this region of the chromosome are indicated; those in the flanking regions (dashed lines) are not shown. Those prefixed with D13 are motif-primed PCR markers (18). Pl1, placental lactogen gene; Ctla2a, ctla2 gene; Tpbp, trophoblast-specific protein gene.



The International Committee on Standardized Genetic Nomenclature for Mice has recommended that the mouse homologue of human PI-6 be designated proteinase inhibitor 3, with a gene Spi3.()

Analysis of the Promoter Region of the Mouse PI-6 Gene

In one of the genomic clones (7), we identified sequences corresponding to the 5` end (exon I) of the mouse cDNA. This clone also contains approximately 650 bp of flanking sequences which are likely to comprise the promoter region of the PI-6 gene. DNA sequence analysis of this region (see Fig. 1) identified a potential TATA box at nucleotide -24 (TATTTAA).

To show that these sequences constitute a functional promoter, a 680-bp fragment (-650 to +31) was subcloned and placed in front of a bacterial chloramphenicol acetyltransferase (CAT) gene in a plasmid derived from pSVTf-CAT(17) . The subcloned fragment contains the 5`-flanking sequences and 31 bp of the 5`-untranslated region of the structural gene (Fig. 1). The resulting plasmid (pPI-CAT) was used to co-transfect BALB/c 3T3 cells along with a vector carrying a G418 resistance gene. G418 resistant clones were pooled, and cell extracts were assayed for CAT activity. Parallel experiments were carried out with a positive control plasmid (pSVTf-CAT) and a negative control plasmid (p2CAT). Assays for CAT activity on cell lysates showed CAT production in cells containing pSVTf-CAT and pPI-CAT, but not in cells containing p2CAT (Fig. 5). These results show that the region immediately adjoining exon I of the PI-6 gene can function as a promoter.


Figure 5: The 5`-flanking region contains a functional promoter. A 680-bp fragment (-650 to +31) of the mouse PI-6 gene was placed in front of the CAT gene in the plasmid pPI-CAT. Clones of BALB/c 3T3 cells carrying this plasmid (or the control plasmids pSVtf-CAT and p2CAT) were derived by co-transfection with a marker plasmid and selection in G418. Extracts from comparable numbers of pooled clones were normalized on protein and assayed for CAT activity by thin layer chromatography analysis of [C]chloramphenicol acetylation. Arrow indicates direction of migration of solvent front; or. indicates origin.



Expression of the Mouse PI-6 Gene in Adults and Embryos

To analyze the expression of the mouse PI-6 gene, we developed a reverse transcriptase-PCR strategy. Tissues and organs were dissected from adult or 15-day-old embryonic mice. Total RNA was prepared and used as template in a reverse transcription reaction primed by oligo(dT). The resulting cDNA was then used for PCR. Each PCR included two sets of primers: one pair to amplify PI-6 sequences and the other pair to amplify glyceraldehyde-3-phosphate dehydrogenase sequences (). The glyceraldehyde-3-phosphate dehydrogenase primers were included as normalization controls and to check the integrity of the RNA/cDNA in tissues lacking PI-6. The PCR products were analyzed on ethidium bromide-stained agarose gels and then transferred to nylon membranes for Southern hybridization using a PI-6-specific oligonucleotide probe.

As shown in Fig. 6, many tissues in the adult produce PI-6, with the highest levels observed in brain and liver. In the embryo (Fig. 7), PI-6 production was noted predominantly in kidney, lung, and yolk sac. Levels were low or absent in the placenta, liver, heart, brain, and muscle. The different patterns of expression between adult and embryo in organs such as liver and brain suggest that PI-6 is developmentally regulated.


Figure 6: Analysis of PI-6 mRNA levels in the adult mouse. Total RNA isolated from the tissues of an adult mouse was used as template for cDNA synthesis with (+) or without (-) reverse transcriptase (RT). A portion of the cDNA reaction was subsequently added to a PCR containing mouse PI-6-specific primers and mouse glyceraldehyde-3-phosphate dehydrogenase-specific primers. Products were separated on a 2% ethidium bromide agarose gel (upper panel), then transferred to a nylon membrane, hybridized to oligonucleotide PB-92, and visualized by autoradiography (lower panel). Lanes 1, heart; 2, brain; 3, spleen; 4, lung; 5, liver; 6, muscle; 7, kidney; and 8, testis.




Figure 7: Analysis of PI-6 mRNA levels in 15-day mouse embryos. Total RNA isolated from the pooled tissues of five 15-day embryos was used as template for cDNA synthesis with (+) or without (-) reverse transcriptase (RT). A portion of the cDNA reaction was subsequently added to a PCR containing mouse PI-6-specific primers and mouse glyceraldehyde-3-phosphate dehydrogenase-specific primers. Products were separated on a 2% ethidium bromide agarose gel (upper panel), then transferred to a nylon membrane, hybridized to oligonucleotide PB-92, and visualized by autoradiography (lower panel). Lanes 1, yolk sac; 2, placenta; 3, liver; 4, heart; 5, gut; 6, kidney; 7, lung; 8, thymus; 9, brain; 10, muscle; and 11, ES cells.




DISCUSSION

We have isolated and characterized cDNA and genomic clones encoding the mouse homologue of human PI-6, which has been designated serine proteinase inhibitor 3 (Spi3) by the International Committee on Standardized Nomenclature for Mice.

Mouse and human PI-6 show 76% similarity at the protein level and are most closely related to the ov-serpins(2) . Of particular interest is the conservation of the residues (Arg-Cys) at the predicted reactive center, suggesting that the two molecules have similar inhibitory properties. This is supported by our observation that, like human PI-6, the mouse molecule interacts with thrombin. The conservation between mouse and human PI-6 also extends to the unusually large number (for serpins) of cysteine and methionine residues in the molecule, particularly in the reactive center region. For human PI-6 we have shown that this feature renders it sensitive to oxidation(7) , and this is also likely to be the case for the the mouse molecule.

Like the rest of the ov-serpins, both mouse and human PI-6 lack discernible amino-terminal signal sequences. Although most of the ov-serpins can be glycosylated and secreted using unconventional signals, there is no evidence of glycosylation in native or recombinant human PI-6(6, 7, 8) , and we have recently shown that the molecule cannot exit the cell via the classical secretory pathway and that artificially glycosylated PI-6 has no proteinase inhibitory activity.()Sequence comparison shows that there is no conservation of the number or position of potential sites for asparagine-linked glycosylation in the human (3 sites) or mouse (1 site) molecules. We consider it unlikely that these molecules are secreted and propose that they fulfill an intracellular role. An intracellular function for serpins is not unprecedented as it is clear that the cytosolic viral serpin CrmA prevents apoptosis by inhibiting an interleukin 1 converting enzyme-like cysteine proteinase(20, 21) .

Analysis of mouse PI-6 gene expression shows that PI-6 is present in many tissues of the adult and embryo. Tissues not surveyed in our previous analysis of human PI-6 mRNA distribution include spleen, thymus, and testis, all of which express PI-6. One obvious difference between the species is the absence of PI-6 from the mouse placenta. There are two possibilities to explain this: PI-6 is only expressed in the placenta at or near term (day 19-20 in the mouse) or PI-6 in the mouse yolk sac (a structure not present in humans) fulfills a function provided by PI-6 in the human placenta. (It is interesting to note that PAI-2 is also found in the human but not the mouse placenta(5) .)

PI-6 levels apparently alter in various organs during development. For example, compared to adults, embryos express relatively low levels of PI-6 in liver and brain and relatively high levels in the kidney. The finding that PI-6 is present in embryonic stem cells suggests that the gene is expressed soon after conception and may play a role in differentiation and development. Further analysis of PI-6 expression in embryos of varying ages and neonates is required to explore this possibility.

Changes in PI-6 levels during development is the first indication of regulated expression of this gene. To date, PI-6 has been noted in a wide variety of tissues and cultured cells, but, unlike PAI-2(5) , alterations in levels in response to growth factors, cytokines, or tumor promoters has not been observed. The 600-bp region flanking the 5` end of the PI-6 structural gene contains elements of the promoter, as shown by its ability to direct transcription of a heterologous gene. Inspection of the sequence reveals a TATA box but no CAAT box upstream of the transcriptional initiation point. However, there is a GC-rich region near the TATA box which may function as an SP1 binding site (22). There are no other discernible motifs for the potential binding of transcription factors, except for an indirect repeat centered at nucleotide -443, and a direct repeat centered on nucleotide -192 which resembles a binding site for members of the C/EBP family. C/EBP is found in tissues such as brain, liver, lung, and skin and is thought to be involved in regulating the balance between cell growth and differentiation(22) .

Soon after the serpin protein family was recognized, interest focused on serpin gene structure. Surprisingly for a family of proteins with such high structural and domain homology, a considerable degree of variation in both the numbers and positions of intron/exon boundaries was noted (reviewed in Ref. 9). This led to two models for serpin gene evolution. The first proposed an ancestral gene containing three introns from which succeeding genes were generated by duplication and intron insertion and deletion. In the second model, an ancestral gene containing at least 22 introns, each marking the borders of distinct domains in the protein, was proposed, from which other serpin genes were generated by duplication and intron deletion(23) .

A number of different serpin gene configurations have now been identified, and there is mounting evidence that the serpin family may be divided into subgroups on the basis of gene position and organization, and that this is a better guide to evolutionary relatedness than amino acid similarity or physiological function. For example, the human protein C inhibitor, corticosteroid binding globulin, -1 proteinase inhibitor, and -antichymotrypsin have similar gene organizations and are clustered on chromosome 14(10) . In the mouse there are two loci on chromosome 12 that contain multiple serpin genes; the Spi1 locus consisting of at least 4 serpin genes and the Spi2 locus of 12 or more genes resembling -antichymotrypsin(24, 25) .

The observation that human PAI-2 and chicken ovalbumin have identical gene structures is consistent with this view and has been used to argue that PAI-2 is the mammalian homologue of ovalbumin(11) . It can also be predicted that the ov-serpins closely resembling ovalbumin and PAI-2 will have similar organizations and locations. Mouse PI-6 is the third potential member of the ov-serpin group for which a gene configuration and localization have been described. As shown in Fig. 8, the structure of the PI-6 gene closely resembles those of PAI-2 and ovalbumin and is distinctly different from other members of the larger serpin family. However, PI-6 lacks an intron present in both ovalbumin and PAI-2 (intron C) and is present on chromosome 13, in a region syntenic with human chromosome 6. This would suggest that human PI-6 is located on chromosome 6, and our recent results confirm this(26) . On the other hand, PAI-2 is localized on human chromosome 18 and mouse chromosome 1(27) , so PI-6 and PAI-2 cannot be part of the same gene cluster. On this basis we propose that PI-6 is not a member of the ov-serpin group, and that it represents a new serpin type that has recently diverged from the ov-serpins. It remains to be seen whether the other ov-serpins (maspin, monocyte/neutrophil elastase inhibitor, squamous cell carcinoma antigen) resemble PAI-2 or PI-6 in gene organization and localization.

  
Table: Oligonucleotides used as probes, sequencing primers, and PCR primers


  
Table: Mapping of the mouse Spi3 (PI-6) gene to chromosome 13 in a (M. musculus M. spretus) F1 M. spretus backcross typing panel



FOOTNOTES

*
This work was supported by the National Heart Foundation and by the National Health and Medical Research Council (Australia). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by 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/EMBL Data Bank with accession number(s) U25843 and U25844.

§
To whom correspondence and reprint requests should be addressed. Tel.: 61-3-989-50316; Fax: 61-3-989-50332.

The abbreviations used are: serpin, serine proteinase inhibitor; PAI-2, plasminogen activator inhibitor; PI-6, proteinase inhibitor 6; PCR, polymerase chain reaction; bp, base pair(s); kb, kilobase(s); CAT, chloramphenicol acetyltransferase.

L. Maltais, personal communication.

F. Scott and P. Bird, unpublished data.


ACKNOWLEDGEMENTS

We thank Lucy Rowe (The Jackson Laboratory, Maine) for analysis and review of the gene mapping data, Dr. Marie Dziadek (Monash University) for assistance with mouse embryo dissections, and Dr. Paul Coughlin for discussions.


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