(Received for publication, March 5, 1997, and in revised form, April 8, 1997)
From the Department of Medicine, Serine proteinase inhibitors (serpins) are
classically regulators of extracellular proteolysis, however, recent
evidence suggests that some function intracellularly. Such
"ovalbumin" serpins include the human proteinase inhibitors 6 (PI-6), 8 (PI-8), and 9 (PI-9), plasminogen activator inhibitor 2, and
the monocyte/neutrophil elastase inhibitor. PI-9 is a potent granzyme B
(graB) inhibitor that has an unusual P1 Glu and is
present primarily in lymphocytes. In a search for the murine equivalent
of PI-9 we screened cDNA libraries, and performed reverse
transcriptase-polymerase chain reaction on RNA isolated from leukocyte
cell lines and from lymph nodes and spleens of allo-immunized mice. We
identified 10 new ovalbumin serpin sequences: two resemble PI-8, two
resemble PI-9, and the remaining six have no obvious human
counterparts. By RNA analysis only one of the two sequences resembling
PI-9 (designated SPI6) is present in mouse lymphocytes while the other
(a partial clone designated mBM2A) is predominantly in testis. SPI6
comprises a 1.8-kilobase cDNA encoding a 374-amino acid polypeptide
that is 68% identical to PI-9. mBM2A is 65% identical to PI-9 and
over 80% identical to SPI6. Although the reactive loops of SPI6 and mBM2A differ from PI-9, both contain a Glu in a region likely to
contain the P1-P1 Serine proteinase inhibitors (serpins) form a superfamily of
proteins that resemble Chicken ovalbumin is the prototype of a branch of the serpin
superfamily (4). These ovalbumin serpins are grouped by amino acid
sequence similarity, and the lack of N- and C-terminal sequences present in Comparisons of gene localization and organization suggest that the
ovalbumin serpins can be subdivided into two groups exemplified by PI-6
and PAI-2. The PAI-2, SCCA-1, SCCA-2, and maspin genes are on human
chromosome 18 (8), whereas the PI-6, PI-9, and MNEI genes are on human
chromosome 6 (16-19). This pattern extends to the mouse where the
PAI-2 gene is on chromosome 1 (20) and the gene encoding the PI-6
homolog, SPI3, is on chromosome 13 (21). Although otherwise identical
in structure, the PI-6 gene lacks an intron present in PAI-2, SCCA-1,
and SCCA-2 (8, 21). By amino acid sequence, PI-8 and PI-9 are more like
PI-6 (13), whereas PI-10 resembles PAI-2 (15).
The physiological roles of most ovalbumin serpins are unknown but MNEI,
maspin, PAI-2, and SCCA may function in tumorigenesis and inflammation.
MNEI has been postulated to control serine proteinases found at
inflammatory sites to prevent damage to surrounding tissue (22), and
maspin is a candidate tumor suppressor (9). Increases in SCCA levels
and release are associated with squamous cell carcinoma (7), while
PAI-2 expression is increased in monocytes during inflammation
(23).
We have recently demonstrated that PI-9 is a potent inhibitor of the
cytotoxic lymphocyte granule serine proteinase, granzyme B (graB), and
have suggested that PI-9 is produced by cytotoxic lymphocytes and other
immune cells to counter misdirected graB released during target cell
destruction (14). To investigate the physiological role of PI-9 we have
decided to study it in the mouse, which has obvious advantages for
studies involving the immune system.
Here we report the identification of 10 new serpin sequences from the
mouse arising from a search for a PI-9 homolog. Among these are two
that are probably PI-8 homologs and two that closely resemble human
PI-9. The remainder represent ovalbumin serpins that have the hallmarks
of functional proteinase inhibitors but have no known human
counterparts. Eight of these new serpin sequences (including one of the
two PI-9 homologs, designated SPI6) were found in activated
lymphocytes. SPI6 was characterized further and shown to be a graB
inhibitor. These results indicate that rodents have a much larger array
of ovalbumin serpins than humans, which probably serves to balance a
larger set of proteinases.
General recombinant DNA methods were
performed according to Ref. 24. cDNA probes were labeled with
[ The mouse cytotoxic T cell line R8 has
been described (25). Mouse allo-immunizations, isolation of spleen and
lymph nodes, and culture of isolated spleen and lymph node cells in
interleukin-2 containing media were as described (26).
Degenerate primers PB117 and
PB118 designed to conserved sequences flanking the PI-6 and ovalbumin
serpin reactive centers were used as described (14). Total RNA was
isolated from cells and tissues as described (27). cDNA was
synthesized from the RNA using Moloney murine leukemia virus reverse
transcriptase and oligo(dT) (24), and amplification and cloning of
products as described (14). Candidate serpin sequences were identified by the presence of the hinge region motif
(Gly-Thr-Glu-Ala-Ala-Ala-Ala(Thr/Ser)) (2) and a conserved
Phe-Cys-Ala-Asp sequence not encoded by the primers.
The SPI6 cDNA clone
was isolated from a mouse day 15 embryo library in The SPI6 cDNA was subcloned into the
EcoRI site of pSVTf (28) so that the 5 The SPI6 cDNA was
modified by PCR to incorporate a hexa-histidine tag immediately after
the initiating methionine. The 1.8-kilobase SPI6 cDNA in For stoichiometric determinations,
10 pmol of graB was incubated with different concentrations of SPI6 at
37 °C in 20 mM Hepes pH 7.4, 100 mM NaCl,
0.05% (w/v) Nonidet-P40 (34). Residual enzyme activity was determined
after 15 min by a two-stage assay using Boc-Ala-Ala-Asp-S-benzyl and
5,5 The SPI6 cDNA cloned in the
P. pastoris expression vector pHILD2 was mutated
using the TransformerTM kit (CLONTECH).
The selection primer 5 Mouse multiple tissue Northern blots
containing 2-µg samples of mRNA from various mouse tissues
(CLONTECH) were probed with a cDNA comprising
the 3 The chromosomal
localization of Spi6 was mapped by analysis of the Jackson
Laboratory (C57BL/B6JEi × SPRET/Ei)F1 × SPRET/Ei backcross
DNA panel (BSS) (37). A partial SPI6 cDNA clone (nucleotides 798-1819) generated by the Erase-a-Base system was used in Southern hybridizations to identify a PvuII polymorphism in genomic
DNA between Mus musculus (C57BL/B6JEi) and Mus
spretus (SPRET/Ei). This was then used to probe a membrane
containing approximately 2 µg of PvuII-digested genomic
DNA from each of the 94 animals in the BSS panel (21). Hybridization
was in 6 × SSC at 68 °C followed by washing in 1 × SSC
at 68 °C. Each DNA sample was scored for the presence or absence of
the M. musculus fragment and the results were
analyzed by L. Rowe at the Jackson Laboratory using the Map Manager
program (38).
We have previously reported that
PI-9 is an inhibitor of graB, and that it is predominantly expressed in
immune cells (14). To assess whether a comparable inhibitor exists in
mouse cells, we incubated a cytosolic extract from R8 cells with
purified human graB. R8 cells are an
interleukin-2-dependent mouse cytotoxic lymphocyte line
that produce granules and are reminiscent of natural killer cells (25).
In many respects they resemble the human YT line which we have shown
produces large quantities of graB and PI-9 (14).
As shown in Fig. 1, incubation of R8 extracts with graB
led to the formation of a 67-kDa complex detected by SDS-PAGE and immunblotting with an anti-graB monoclonal antibody. This resembled the
SDS-stable complex formed between graB and PI-9, which is characteristic of serine proteinase-serpin interactions (2). In
addition, immunblotting with anti-PI-9 antibodies weakly detected a
42-kDa protein in R8 extracts (data not shown). From these experiments we concluded that a mouse graB inhibitor and homolog of PI-9 is present
in R8 cells.
In a parallel approach we used RT-PCR to identify ovalbumin serpin
sequences present in mouse immune tissue. Given its presence in
activated lymphocytes (14), it is conceivable that PI-9 or its homologs
are primarily produced during an immune response. To allow for this, we
took cells from the popliteal lymph nodes and spleens of allo-immunized
mice. Some of these lymph node and spleen cells were then cultured for
1-2 weeks in the presence of interleukin-2, which selectively
activates and promotes the proliferation of cytotoxic lymphocytes
(in vitro stimulated). R8 cells and a long-term (>6 months)
culture of mouse spleen cells passaged in high levels of interleukin-2
were also examined. The latter cells (designated NK) exhibit natural
killer-like, non-MHC restricted cytotoxic
function.2
RNA was prepared from each of these sources and used as a template for
cDNA production and PCR. The PCR primers were degenerate oligonucleotides designed to ovalbumin serpin sequences that have been
previously used to identify PI-9 (14). These amplify the serpin
reactive center loop and candidate sequences are identified by
conserved residues inside the primer-binding sites. In our hands this
primer pair has amplified PAI-2, MNEI, PI-6, PI-8, PI-9, and heparin
cofactor II from various human
tissues.3
As shown in Fig. 2, RT-PCR of the mouse cells and tissue
identified nine serpin sequences (SPI3, SPI6, mNK10, mAT2, mR86, mNK9,
mNK13, mNK21, and mNK26). One of these (SPI3) is the previously described mouse homolog of PI-6 (21). The relative frequency that these
new sequences appeared is indicated in Table I. All but
one (mNK26) appeared more than once, in independent clones arising from
different samples. All of the new sequences possessed the serpin
proximal hinge motif Gly-Thr-Glu-Ala-Ala-Ala-Ala(Thr/Ser) (39) inside
the upstream-primer site, indicating that they are functional
proteinase inhibitors. In addition, most possessed a Phe-Cys-Ala-Asp
motif adjacent to the downstream primer site. The latter motif is found
in the ovalbumin serpin subgroup that comprises PI-6, PI-8, PI-9, and
MNEI (see Fig. 2).
Table I.
Frequency of new serpin sequences in mouse tissue observed by RT-PCR
Cellular
Cytotoxicity Laboratory,
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
bond. SPI6 produced in
vitro using a coupled transcription/translation system formed an
SDS-stable complex with human graB and did not interact with trypsin,
chymotrypsin, leukocyte elastase, pancreatic elastase, thrombin, or
cathepsin G. Recombinant SPI6 produced in a yeast expression system was used to examine the interaction with human graB in more detail. The
second-order rate constant for the interaction was estimated as 8 × 104 M
1 s
1, and
inhibition depended on the Glu in the SPI6 reactive center. The SPI6
gene was mapped to the same region on mouse chromosome 13 as
Spi3, which encodes the murine homolog of PI-6. We conclude that even though their reactive centers are not highly conserved, SPI6
is a functional homolog of PI-9, and that the regulation of graB in the
mouse may involve a second serpin encoded by mBM2A. Our identification
of multiple sequence homologs of PI-8 and PI-9, and six new ovalbumin
serpins, is consonant with the idea that the larger set of granule and
other proteinases known to exist in the mouse (compared with human)
is balanced by a larger array of serpins.
1-proteinase inhibitor in
structure (1). The majority regulate proteinase activity in
extracellular processes such as fibrinolysis, blood coagulation,
complement activation, and tissue remodeling (2). Serpins generally
contain an exposed C-terminal loop in which the reactive center
P1-P1
residues define the inhibitory
specificity of the molecule. Inhibition of proteolysis is achieved by
the formation of essentially irreversible 1:1 stoichiometric complexes
between serpins and cognate proteinases (2, 3).
1-proteinase inhibitor. They include
plasminogen activator inhibitor (PAI-2
(5)),1 monocyte/neutrophil elastase
inhibitor (MNEI (6)), squamous cell carcinoma antigens (SCCA-1 and 2 (7, 8)), maspin (9), proteinase inhibitor 6 (PI-6 (10-12)), proteinase
inhibitor 8 (PI-8 (13)), proteinase inhibitor 9 (PI-9 (13, 14)), and
proteinase inhibitor 10 (PI-10 (15)).
General Methods
-32P]dATP (DuPont NEN) using the random hexamer
method (Prime-A-Gene System, Promega).
gt10
(CLONTECH). The mBM2A and mBM17 clones were
isolated from a mouse bone marrow library in
gt11
(CLONTECH). Approximately 4 × 105
plaques were screened in each case at low stringency using a partial
cDNA clone of human PI-9 (14). Membranes were hybridized at
32 °C in 50% formamide, 5 × SSPE, 0.5% SDS, and 5 × Denhardt's, then washed in 2 × SSC and 0.1% SDS at 45 °C.
Positive plaques were purified and the EcoRI inserts were
subcloned into the EcoRI site of pUC118 or pBluescript II
KS
(Stratagene). Both strands of each cDNA were
sequenced using the Sequenase II system (Amersham) either on
overlapping clones generated by directed exonuclease III deletion
(Erase-a-Base system, Promega), or by an oligonucleotide walk strategy.
Sequences were assembled and analyzed using the DNASIS programs
(Hitachi).
end of the clone was
closest to the T7 promoter. Purified plasmid DNA was linearized with
SalI prior to transcription/translation. For control
production of human PI-6, pSVPTI/P (11) was linearized with
SalI. Labeled protein was produced in a coupled
TNTTM Wheat Germ Extract system (Promega) using T7
polymerase and incorporating [35S]methionine (DuPont
NEN). Translation products were analyzed by SDS-PAGE (29) or native
PAGE (30) followed by fluorography (AmplifyTM, Amersham).
For analysis of complex formation, 2 µl of the translation products
were incubated at 37 °C for 30 min with 2 ng of the appropriate proteinase. Purification of active graB was as described (31). Thrombin
was purified as described (32), chymotrypsin, trypsin, leukocyte
elastase, pancreatic elastase, and cathepsin G were purchased from
Sigma.
gt10
was used as a template in a PCR primed by
5
-TCTGCCATCATGCATCATCATCATCATCATAATACTCTGTCTGAAGGAAATGG-3
(sense) and
5
-TTATGGAGATGAGAACCTGCCACA-3
(antisense). Amplification, cloning,
verification, and expression of the modified SPI6 cDNA in
Pichia pastoris was as described previously for PI-6 and
PI-9 (14, 33).
-dithiobis(nitrobenzoic acid) (35). The rate of inhibition of graB
by SPI6 was determined by incubating equimolar enzyme and inhibitor at
37 °C, and determining residual activity periodically (33, 36). The
second-order rate constant was calculated as described (33).
-CGGTGAGCATGCAGACCTTCAAC-3
removes a unique
XbaI in pHILD2. The mutagenic primer
5
-GCCATCATAGCATTTTGCTGTGCC-3
substitutes 337Glu in the
SPI6 sequence with Ala. Following verification by DNA sequencing, the
mutated SPI6 cDNA was subcloned from pHILD2 to pSVTf for use in the
coupled transcription/translation system.
-untranslated region of the SPI6 cDNA (nucleotides
1035-1819). Hybridization was carried out at 42 °C in 5 × SSPE, 10 × Denhardt's, 0.1% SDS, and 50% formamide. Membranes were then washed in 0.1 × SSC and 0.1% SDS at 65 °C and
exposed to x-ray film. The membrane was subsequently stripped and
re-probed under the same conditions with the mBM2A cDNA.
Identification of GraB Binding Activity and Multiple Serpin
Sequences in Mouse Immune Cells
Fig. 1.
Identification of a graB binding activity in
mouse cells. Cytosolic extracts were prepared from R8 cells and
incubated in the absence or presence of 10 ng of purified human graB.
Samples were boiled and reduced prior to electrophoresis on a 12%
SDS-polyacrylamide gel, then immunoblotted with a graB monoclonal
antibody. The arrow shows the position of the SDS-resistant
complex containing graB.
[View Larger Version of this Image (45K GIF file)]
Fig. 2.
New mouse serpin sequences identified by
cDNA library screening and RT-PCR. Upper panel shows the
inhibitory region/reactive center loop sequences of the previously
characterized human ovalbumin serpins PAI-2 (5), SCCA-1 (7), SCCA-2
(8), maspin (9), PI-6 (11), PI-8 (13), PI-9 (13, 14), PI-10 (15), and MNEI (6), and the prototype serpin sequence,
1-antiproteinase (1). Lower panel shows the
mouse serpin sequences identified in this study. SPI3 is the mouse
homolog of PI-6 (21). Reactive center bonds
(P1-P1
) are indicated by the vertical
arrow. Dashes indicate gaps introduced for optimum sequence
alignment. The sequences used for the design of the degenerate PCR
primers are indicated by the horizontal arrows above the
PAI-2 sequence.
[View Larger Version of this Image (70K GIF file)]
Sequence
Source of RNA
R8 cells
NK
cells
Spleen
Popliteal
Spleen in vitro
Popliteal
in vitro
SPI3
26 /45
23
/35
49 /69
15 /35
4 /23
32 /36
SPI6
16 /45
4
/35
3 /69
2 /35
3 /23
2 /36
mBM2A
0 /45
0
/35
0 /69
0 /35
0 /23
0 /36
mBM17
0 /45
0
/35
0 /69
0 /35
0 /23
0 /36
mNK10
0 /45
1
/35
3 /69
10 /35
3 /23
0 /36
mAT2
0 /45
0
/35
0 /69
0 /35
2 /23
0 /36
mR86
3 /45
0 /35
2
/69
4 /35
1 /23
1 /36
mNK9
0 /45
2 /35
1 /69
2
/35
2 /23
0 /36
mNK13
0 /45
2 /35
11 /69
1
/35
5 /23
1 /36
mNK21
0 /45
2 /35
0 /69
1
/35
3 /23
0 /36
mNK26
0 /45
1 /35
0 /69
0
/35
0 /23
0 /36
Comparison and alignment of the mouse sequences with the human serpin sequences indicated that only one of the novel sequences (mNK10) has a clear human counterpart. This is probably a homolog of PI-8 since it shows 73% identity in the variable region between the proximal hinge and the Phe-Cys-Ala-Asp motif, and it has an identical P1 Arg residue.
Surprisingly, sequence comparisons alone revealed no obvious homolog of
PI-9. However, the second most frequent sequence arising from R8 cells
after SPI3 was SPI6, and this sequence was identified in all the other
samples. Although there is only 39% identity between the SPI6 and PI-9
variable regions, inspection and alignment of the SPI6 reactive center
with the other serpins suggested that a Glu would be at, or close to,
the P1-P1 bond. Since (i) a Glu at this
position is a unique feature of PI-9 (14) and is crucial for graB
inhibition,4 (ii) SPI6 sequences arose most
frequently in R8 cells which also contain a graB-binding function, and
(iii) the other serpin (SPI3) observed in R8 cells does not interact
with graB, we hypothesized that SPI6 is a functional homolog of
PI-9.
To isolate a
complete SPI6 cDNA, mouse bone marrow and day 15 embryo cDNA
libraries were hybridized at low stringency to a partial human PI-9
cDNA. Isolates from the bone marrow library included several
incomplete SPI3 and SPI6 clones, as well as two new serpins not already
identified by RT-PCR (mBM2A, mBM17; Fig. 2). Analysis of
these partial clones suggested that mBM17 is another homolog of PI-8,
being 64% identical in the variable region with an identical
P1 Arg residue (and over 80% identical to mNK10). The
clone mBM2A is highly related to SPI6 and probably represents a second
PI-9 homolog in the mouse (see Fig. 3B and
"Discussion" below).
Three clones were isolated from the mouse embryo library. Two
corresponded to the characterized serpins SPI3 and mouse
1-antitrypsin, while the third clone contained a
1.8-kilobase insert encoding SPI6. (The designation serine proteinase
inhibitor 6 (SPI6; gene Spi6) has been allocated by the
International Committee on Standardized Nomenclature for Mice.)
Sequencing of the SPI6 cDNA predicted a protein of 374 amino acids
(Fig. 3A). The coding region is flanked by a short
5-untranslated region containing two termination codons (one in frame
with the first methionine), and is followed by a 653-base pair
3
-untranslated region which includes a polyadenylation signal at
nucleotides 1684. However, the cDNA extends for a further 135 base
pairs and has no poly(A) tail, suggesting that Spi6
transcription can continue past this first polyadenylation signal.
Transcriptional termination and polyadenylation might then be directed
by another signal not represented in this clone. Two polyadenylation
signals would allow transcriptional termination to occur at either of
two points, and may explain the presence of two SPI6 transcripts
detected by Northern blotting (see Fig. 5A).
Sequencing of the mBM2A partial clone revealed a serpin highly related
(85%) to SPI6 (Fig. 3B). Compared with other serpins, SPI6
and mBM2A are most like PI-9 (68 and 65% identity, respectively). SPI6
and mBM2A show greatest identity to the PI-6-like serpins suggesting
they are members of this ovalbumin serpin subgroup. For example, SPI6
shows 55% identity with SPI3, 48% with MNEI, and 55% with PI-8 (Fig.
4), compared with 43, 41, and 34% for the SCCAs, PAI-2,
and maspin, respectively. It shows 37% overall identity with the
cowpox serpin cytokine response modifier A (a viral inhibitor of graB)
rising to 41% through the reactive center loop.
The reactive center proximal hinge regions of SPI6
(Gly326-Thr-Glu-Ala-Ala-Ala-Ala-Ser-Ala) and mBM2A
(Gly139-Thr-Glu-Ala-Ala-Ala-Ala-Ser-Ala) are conserved
according to the consensus sequence for inhibitory serpins (39). This
predicts that both are functional proteinase inhibitors. Alignment
(without introducing deletions) of the SPI6 and mBM2A polypeptide
sequences with the various human and mouse ovalbumin serpins predicts
that the P1-P1 residues in the reactive center
loop are Cys340-Ala341 and
Cys153-Ala154, respectively. However, the
optimal alignment suggests Phe338-Cys339 or
Tyr151-Cys152, respectively (see Figs. 2 and
3B). The presence of Cys residues at or near the reactive
center suggests that SPI6 and mBM2A may be oxidation sensitive, and is
consistent with an intracellular localization and role. PI-6, PI-8,
PI-9, and MNEI also have cysteines in their reactive center loops, and
alkylation of MNEI and PI-6 destroys activity (6, 11).
[35S]Methionine-labeled SPI6 was produced in vitro using a coupled transcription/translation system. The translation products were analyzed by SDS-PAGE and fluorography. As predicted from the sequence, a protein of approximately 42 kDa was produced from the SPI6 cDNA (Fig. 5).
Labeled SPI6 was then tested for complex formation with graB and a number of other serine proteinases. A feature of serpin-serine proteinase interactions is the formation of a stable complex that is not dissociated by SDS (2). Addition of labeled SPI6 to purified graB resulted in the formation of a 67-kDa complex that was apparent following reduction, boiling, and electrophoresis in SDS (Fig. 5). Human PI-9, which forms an SDS-stable complex with graB (14), was produced in the same system and incubated with graB. As expected, a 67-kDa graB-PI-9 complex was observed (Fig. 5).
SPI6 did not complex with any of the following proteinases: trypsin, chymotrypsin, leukocyte (neutrophil) elastase, pancreatic elastase, thrombin, or cathepsin G (data not shown). Similar results were obtained by native PAGE (data not shown). These experiments suggest that SPI6 is a likely mouse homolog of PI-9.
Inhibition of Human GraB by SPI6Hexa-histidine tagged recombinant SPI6 was produced in a yeast expression system and purified by nickel affinity chromatography. As predicted from the cDNA sequence, the purified recombinant protein had a molecular mass of 42 kDa (not shown).
Complex formation between a serpin and proteinase follows second-order
kinetics, and association rate constants of 105 to
107 M1 s
1 represent
physiologically significant interactions (3). The reaction
stoichiometry is usually equimolar and initial formation of a Michaelis
complex is followed by the formation of a kinetically stable (locked)
tetrahedral complex (2, 3). To examine the interaction of SPI6 and graB
in more detail, we first established the stoichiometry of the reaction
by titrating a fixed amount of graB against varying amounts of
inhibitor and measuring residual proteolytic activity (data not shown).
The 1:1 ratio observed is typical of a serine proteinase-serpin
interaction (2). The association rate constant (ka)
for complex formation was calculated as 8 ± 0.8 × 104 M
1 s
1. This is
just outside the range for physiologically important serpin-proteinase
interactions (2, 3), and is somewhat lower than the constant for the
graB and PI-9 interaction (1.7 × 106
M
1 s
1 (14)). This poorer than
expected inhibition may simply reflect the different species origin of
the serpin and proteinase (see "Discussion").
A unique feature of PI-9 is the presence of an acidic residue (Glu) at the P1 position, which is consistent with its role as an inhibitor of a proteinase cleaving after acidic residues (Asp or Glu). Substitution of the P1 Glu in PI-9 dramatically lowers inhibitory activity against graB.4 Because SPI6 and mBM2A have shorter reactive center loops than PI-9 and other ovalbumin serpins, it is difficult to identify the likely P1 residues by sequence alignment alone. However, both SPI6 and mBM2A have only one acidic residue (Glu) in their reactive center loops, and it is likely that this is important for inhibition of graB. To test this we used site-directed mutagenesis to produce a derivative of SPI6 in which Glu337 has been substituted by Ala. This derivative was produced in the coupled transcription/translation system and in the yeast expression system. As shown in Fig. 5, labeled mutant protein did not form a detectable complex with graB on SDS-PAGE. In addition, although we were able to produce and purify equivalent amounts of the mutant from the yeast system (compared with SPI6), we were unable to demonstrate any graB inhibitory activity (data not shown). We conclude that Glu337 is crucial for the inhibitory activity of SPI6, and that it may represent the P1 residue.
Tissue Distribution of SPI6 and mBM2A mRNATo determine
the tissue distribution of SPI6 and mBM2A, a membrane containing
mRNA from mouse brain, heart, kidney, liver, lung, skeletal muscle,
spleen, and testis was sequentially hybridized to
32P-labeled SPI6 and mBM2A cDNA probes. With an SPI6
3-untranslated region probe, two transcripts of approximately 2.4 and
3.6 kilobases were detected in most tissues, but predominantly in
heart, lung, spleen, and kidney. The highest levels were in lung and
spleen (Fig. 6). The nature of the two transcripts is
unknown but they may arise by differential splicing or (as discussed
above) differential transcriptional termination.
When the same membrane was stripped and hybridized to the mBM2A cDNA, the most prominent signal was obtained in testis (Fig. 6). Here a single 1.7-kilobase transcript was observed, that was not detected in other tissues. Fainter bands of identical size, distribution, and relative intensities as those seen with the SPI6 probe were also observed. These are likely to represent cross-hybridization of the mBM2A probe with SPI6 transcripts. We conclude that while mBM2A expression is essentially restricted to testis, SPI6 expression is somewhat broader.
Chromosomal Localization of Spi6The PI-6, human MNEI, and PI-9 genes co-localize on human chromosome 6p25 (17, 18, 20, 40), while the PAI-2, SCCA, and maspin genes are clustered on human chromosome 18q21 (8). If SPI6 is a new member of the PI-6 family it is likely that the gene will localize to the same region on chromosome 13 as the gene encoding the mouse PI-6 homologue, SPI3.
The chromosomal localization of Spi6 was determined by
hybridization of a partial SPI6 cDNA to a panel of DNAs from
interspecific backcross mice (37). The SPI6 fragment (798-1819 base
pairs) detected a PvuII polymorphism between
M. musculus (C57BL/BJEi) and M.
spretus (SPRET/Ei) and was then hybridized to PvuII
digested genomic DNA from each of the 94 animals derived from a
(M. musculus × M. spretus)F1 × M. spretus backcross. After scoring each sample for the
presence or absence of the M. musculus fragment,
Spi6 was localized to mouse chromosome 13 in the same region
as mouse Spi3 (Fig. 7). Spi3 and
Spi6 could not be separated by this analysis, indicating a
distance between them of 0-3.8 centimorgan. Data in Table
II show the recombination frequencies for the nearest linkage markers for Spi6. Spi6 is localized approximately 20 centimorgans from the centromere of chromosome 13 between the placental
lactogen 1 gene (3.2 centimorgans proximal) and the motif-primed PCR
marker D13Bir9 (1.1 centimorgans distal).
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In this study we report the identification of 10 previously unknown serpin sequences in the mouse. Most of the new sequences were identified by RT-PCR using primers specifically designed to amplify ovalbumin serpin inhibitory regions, and sequence analysis shows that each new serpin has a Phe-Cys-Ala-Asp motif in the distal portion of this region that is present in most human ovalbumin serpins but not in other members of the serpin superfamily. Thus these new mouse serpins are probably members of the ovalbumin serpin subgroup. Based on the presence of consensus proximal hinge motifs each of these serpins is likely to be a proteinase inhibitor, but in the absence of functional data possible targets of these serpins cannot easily be predicted. It should be noted, however, that recent studies have shown that serpins are capable of inhibiting more than one class of proteinase (34, 41). Hence the cognate proteinases of these mouse serpins may include serine proteinases and cysteine proteinases (caspases or papain-like cathepsins).
Even though our search for mouse ovalbumin serpins was largely
restricted to activated lymphocytes, the number of new sequences identified suggests that the ovalbumin serpin family in the mouse is
much larger than its human counterpart. The existence of many more
serpins in the mouse has already been suggested by the description of
two gene clusters on mouse chromosome 12 that have only single human
gene equivalents (42, 43). These clusters have almost certainly arisen
by multiple gene duplications and encode serpins resembling
1-antiproteinase (Spi1) or
1-antichymotrypsin (Spi2). It is likely that,
although closely related, individual members of each cluster have
different inhibitory capacities. The family of mouse ovalbumin serpins
identified here have at least three human counterparts (PI-6, PI-8, and
PI-9) and almost certainly also target a diverse range of proteinases.
The fact that Spi6 maps to the same chromosomal region as
Spi3 suggests that a serpin gene cluster comprising some of
the mouse ovalbumin serpins is present on chromosome 13, and that SPI6
is a member of the PI-6 ovalbumin serpin subfamily and not the PAI-2
group. This is supported by sequence comparisons which show that SPI6
more closely resembles mouse PI-6 (SPI3, 55%) than mouse PAI-2 (40%).
(Fig. 4 shows a comparison of SPI6 with the other members of the PI-6
group.) It remains to be seen whether Spi6 resembles
Spi3 in gene organization and lacks an intron present in
PAI-2, SCCA-1, SCCA-2, and ovalbumin (21).
The larger array of inhibitory serpins in the mouse suggests that rodents have many more proteinases that must be regulated. This is illustrated by a comparison of the granule proteinase complements of cytotoxic lymphocytes in humans and rodents. In humans there are five granule serine proteinases whereas in rodents there are at least nine (44). It is conceivable that some of the new serpins we have identified that lack obvious human counterparts are involved in regulating granule serine proteinases specific to the mouse. Since most of these new serpins are likely to be lymphocyte products it is probable that other mouse leukocytes have a distinct ovalbumin serpin complement, perhaps with novel members not identified here. For example, monocytes and neutrophils should have one or more counterparts of PAI-2 and MNEI. At present only one mouse homolog of PAI-2 is known (20). Due to the primer design, we may not have amplified non-inhibitory serpin sequences so it is also conceivable that non-inhibitory mouse ovalbumin serpins await discovery.
An interesting observation arising from this work is the apparent existence of multiple mouse homologs of at least two human ovalbumin serpins. Specifically, the clones mBM17 and mNK10 are probably homologs of human PI-8, while the clones mBM2A and SPI6 are homologs of human PI-9. It is possible that the different homologs play distinct roles in the mouse. For example, SPI6 and mBM2A may both regulate graB, but do so in separate contexts. This is supported by the fact that mBM2A and SPI6 have a different tissue distribution. Alternatively, these two serpins may regulate related but distinct proteinases within the mouse. Similarly, mBM17 and mNK10 probably have different roles since a different tissue distribution is implied by the presence of mNK10, and absence of mBM17, in lymphocytes.
On the basis of its properties and distribution, we have proposed that PI-9 functions to protect cytotoxic lymphocytes and other immune system components, such as antigen-presenting cells, from mis-directed graB (14). It is most likely that SPI6 is the true PI-9 homolog because (i) it has over 68% sequence identity with PI-9; (ii) it is also a graB inhibitor; (iii) inhibition depends on a glutamic acid residue in the reactive loop; and (iv) like PI-9 it is present in lymphocytes and spleen cells. However, SPI6 shows a broader tissue distribution than PI-9, being present in heart, lung, and kidney. At present we do not know whether this indicates an additional function for a graB inhibitor in the mouse, or whether it simply represents a different distribution of antigen-presenting or cytotoxic (CD8+ and CD56+) cells in mouse tissues.
Although we have been unable to do functional studies with mBM2A, its expression pattern (predominant in testis, absent in lymphocytes and spleen cells) rules it out as a direct homolog of PI-9. If mBM2A is a graB inhibitor, its presence in mouse testis may be related to the immune-privileged status of certain cells within the testis. It has been shown recently that Sertoli cells in the mouse testis express Fas ligand, and can induce apoptosis of cytotoxic lymphocytes that come in contact with them (for review, see Ref. 45). It may be that mouse Sertoli cells also produce a graB inhibitor (mBM2A) to protect against the granule-mediated arm of the CL killing machinery. Alternately, mBM2A may regulate a different proteinase to graB. Recent evidence suggests that there is more than one serine aspase in rodents.
One problem with the proposal that SPI6 and PI-9 are homologs is the
relative inefficiency with which SPI6 inhibits human graB, but this may
be a consequence of the lower than expected sequence conservation
(39%) in the SPI6 and PI-9 inhibitory loops. Specifically, the SPI6
loop is two amino acids shorter than PI-9, there is an aromatic amino
acid near the P1-P1 residues in SPI6 but not
in PI-9, and the putative P1 glutamic acid residue is much
closer to the proximal hinge in SPI6 than in PI-9. Obviously these
differences may reflect structural variations in the active sites of
mouse and human graB, and in their substrate specificity. Formal
resolution awaits the availability of practical quantities of purified
mouse graB to test with SPI6 and PI-9. We expect that SPI6 will inhibit
mouse graB more efficiently than human graB.
In conclusion, we have provided evidence for a large family of ovalbumin serpins in mouse lymphocytes and marrow, and the existence of multiple homologs of two human intracellular serpins. The distribution and properties of these new serpins suggests that rodents possess additional proteolytic systems that contribute to immune function. This might reflect differing evolutionary pressures placed on rodent and human immune systems, and suggests that caution should be exercised in extrapolating results from rodent models to humans.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U96700[GenBank], U96701[GenBank], U96702[GenBank], U96703[GenBank], U96704[GenBank], U96705[GenBank], U96706[GenBank], U96707[GenBank], U96708[GenBank], U96709[GenBank].
We thank J. Rose (University of Melbourne) for the BSS membranes, Lucy Rowe (The Jackson Laboratory) for analyzing the chromosomal localization data, and the anonymous reviewer who in the early stages of the work encouraged us to consider that the similarities between SPI6 and PI-9 outweighed the differences.