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J. Biol. Chem., Vol. 277, Issue 44, 42028-42033, November 1, 2002
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From the
Received for publication, July 15, 2002, and in revised form, August 17, 2002
The human ov-serpin monocyte neutrophil elastase
inhibitor (MNEI) is encoded by a single gene SERPINB1. It
is a highly efficient inhibitor of neutrophil granule proteases. Four
murine genes with high sequence identity with MNEI were identified and
fully sequenced, and these were named EIA, EIB, EIC, and EID. EIA, EIB
and EIC showed the same seven-exon gene structure as
SERPINB1. However, EIC included an additional,
alternatively spliced, exon due to the insertion of an endogenous
retrovirus-like sequence. EID lacked several exons and is a
pseudogene. Reverse transcriptase-PCR showed that EIA, like MNEI, is
expressed at high levels in many tissues. EIB is mainly expressed in
brain, and EIC was only expressed as splicing variants unlikely to
encode a functional serpin. Upon incubation with serine proteases, EIA
formed inhibitory covalent complexes with pancreatic and neutrophil
elastases, cathepsin G, proteinase-3, and chymotrypsin, as previously
shown for MNEI, whereas EIB was only able to do so with cathepsin G. According to the new serpin nomenclature, the genes encoding EIA, EIB,
EIC, and EID will be called Serpinb1,
Serpinb1b, Serpinb1c, and
Serpinb1-ps1. These data demonstrate that the four murine
homologs of MNEI have met different evolutionary fates, and that EIA is
the mouse ortholog of MNEI.
Serpins (serine protease inhibitors) are a superfamily of
~45-kDa proteins with a highly conserved tertiary structure. Serpins regulate important intracellular and extracellular proteolytic events,
including apoptosis, complement activation, fibrinolysis, and blood
coagulation (1, 2).
Serpins inhibit proteases by a suicide substrate inhibition mechanism,
whereby the protease reactive center interacts with a bait cleavage
site between the P1 and P1' residues of the reactive center loop of the
serpin (3). The two molecules form a 1:1 stoichiometric intermediate,
which is stabilized into a covalent inhibitory complex. Fluorescence
energy transfer experiments have shown that the stable inhibitory
complex involves full insertion of the cleaved proximal reactive center
loop (RCL)1 of the serpin
into According to a recent phylogenetic analysis (8), human serpins have
been classified in nine clades. Clade B, also called ov-serpins
(ovalbumin-related serpins), is the second largest clade in humans with
13 members identified so far. OV-serpins share a high degree of
sequence identity and conserved exon-intron patterns. They differ from
other serpin clades by the lack of N- and C-terminal extensions, the
lack of a cleavable hydrophobic signal sequence for secretion and the
presence of other signature sequence motifs (9). Recent studies of
exon-intron structures indicate that serpins are likely to have evolved
by massive intron insertion and show that ov-serpins form an
evolutionary distinct clade (10, 11). Furthermore, in contrast to other
serpin clades, ov-serpins lack signal sequences for secretion and are
found in the cytoplasm (9) and, to a lesser extent, on the cell surface (12) and in the nucleus (13). Human ov-serpin genes are clustered in
two loci, 6p25 and 18q21, and fall into two subgroups based on the
number of exons (14). The eight-exon ov-serpins are only found on
chromosome 18, whereas the seven-exon ov-serpins are found on both loci.
The seven-exon ov-serpin, human monocyte neutrophil elastase
inhibitor (MNEI, SERPINB1), is one of the most efficient
inhibitor of the neutrophil granule proteases, which include neutrophil elastase, proteinase-3 (PR3), and cathepsin G (CatG) (15-17). These neutrophil granule proteases are directly responsible for the killing
of phagocytosed pathogens (18), but, conversely, an excessive release
of neutrophil proteases in the extracellular milieu induces tissue
damage (19) and impairs the clearance of apoptotic cells in chronic
inflammatory diseases (20). In an animal model of chronic lung
inflammation, aerosolized recombinant human MNEI reduced the severity
and the extent of the elastase-induced tissue damage (21). The
sustained activity of MNEI after instillation supports its potential
use as a therapeutic for inflammatory pulmonary diseases driven by
excess of neutrophil-derived proteases, such as cystic fibrosis and
chronic pulmonary disease. To better understand the biological role of
MNEI, we describe here the cDNA cloning, the gene structure, and
biological characterization of the putative murine ortholog of MNEI
together with three paralogs.
cDNA Data Base Mining--
Using human MNEI cDNA
sequence, mouse dbEST data base was searched with BLAST
(www.ncbi.nlm.nih.gov/BLAST/), and matches were further analyzed
using DNAStar software for PC (DNAStar Inc., Madison, WI). Selected
clones with high homology were obtained from the I.M.A.G.E.
Consortium (22) and sequenced.
Southern Blot Analysis--
Based on the cDNA sequence of
EIA (AF426024) and the gene structure of MNEI (SERPINB1),
two 32P-labeled probes in the exon 7 coding region and
3'-untranslated region were generated as described previously (23).
Mouse genomic DNA was digested with restriction enzymes, separated on
0.8% agarose gels, and transferred onto nitrocellulose membranes.
Membranes were hybridized, washed, and autoradiographed as described
previously (23).
Genomic DNA Sequencing and Analysis--
A genomic lambda phage
library from 129/SvJ mice, a generous gift from Dr. Michael Carroll
(Center for Blood Research, Boston, MA), was screened with a
32P-labeled EIA exon 7 probe. Lambda clone #1 containing
EIA was digested with SacI, and lambda clone #3 containing
EIB was digested with BamHI. Fragments were subcloned in
pBluescript (Stratagene) and sequenced. The sequence data of EIA and
EIB from these subclones were fragmentary, and mouse PAC clones from
library RPCI21, constructed using female 129/SvevTACfBr mouse genomic
spleen, were screened with EIA- and EIB-specific primers. PAC clone
507G8 was shown to contain EIA and two apparent paralogs, EIC and EID.
PAC clone 672N8 contained EIB. Introns were cloned from PAC 507G8,
using primers based on cDNA or genomic sequences from
CeleraTM or GenBankTM databases. All PCR
fragments were amplified with Turbo Pfu polymerase (Stratagene), cloned in pSTBlue-1 or pT7Blue-2 (Novagen), and sequenced
in both directions using an "oligonucleotide walk" strategy at the
Molecular Biology Core Facilities of the Dana Farber Cancer Institute
(Boston, MA). These PCR fragments were designed to overlap widely
within exons to build the contig without ambiguity on the origin of the fragments.
RT-PCR and Rapid Amplification of cDNA Ends--
Mouse
brain, heart, liver, lung, spleen, testis total RNA, and bone marrow
and pancreas mRNA were obtained from Clontech
(Palo Alto, CA). Five micrograms of total RNA or 1 µg of mRNA
were reverse-transcribed in a 40-µl reaction using Superscript II and
oligo(dT) primers (Invitrogen, Carlsbad, CA). Identical control
reactions were performed without reverse transcriptase to rule out
genomic DNA contamination. mRNA expression levels of the
"housekeeping" gene
Specific primers for each of the four serpin cDNA were used with
Platinum Taq DNA polymerase (Invitrogen) in 50-µl PCR
reactions for 30 and 35 cycles. The primer sequences and their
positions are shown in Table I. Levels of
expression were analyzed by densitometry on ethidium bromide-stained
agarose gels. RACE reactions were performed as described
previously (25), using 5'/3'-RACE kit (Roche Molecular Biochemicals)
and heart total RNA (Clontech).
In Vitro Transcription/Translation--
The complete
cDNA coding sequences of EIA (AF426024) and EIB (AF426025) were
subcloned into pET17b (Novagen). The proteins were synthesized by
in vitro transcription and translation in the presence of
[35S]methionine for 2 h at 37 °C using an
Escherichia coli T7 S30 extract system for circular DNA (Promega).
Inhibitory Complex Formation with Proteinases--
Human
neutrophil elastase (hNE), and porcine pancreatic elastase (pPE) were
from Elastin Products (Owensville, MO); cathepsin G (hCatG) and
proteinase-3 (hPR3) from human neutrophils were from Athens Research
and Technology (Athens, GA); bovine chymotrypsin (bChy) was from Sigma
(St. Louis, MO). They were stored in 1 mg/ml aliquots at
The ability of recombinant EIA and EIB to form inhibitory complexes
with proteases was assessed by incubating 2.5 µl of the in
vitro translation reaction with different concentrations of proteases in a final volume of 20 µl phosphate-buffered saline (pH
7.4). All the reactions were prepared on ice and incubated for 5 min at
37 °C. Where indicated, EIB was incubated with proteases for up to
30 min. Samples were chilled on ice and acetone-precipitated. Pellets
were resuspended in 1× NuPAGE LDS sample buffer (Invitrogen), boiled
for 3 min, loaded onto 10% Bis-Tris NuPAGE gels, and separated under
reducing conditions in MES buffer. 35S-Labeled proteins
were detected in dried gels by phosphorimaging (Storm 860 PhosphorImager, Molecular Dynamics, Sunnyvale, CA) and analyzed
using the ImageQuant software (Molecular Dynamics).
Gene Structure of the Mouse Homologs of MNEI--
In preliminary
screening of the dbEST data base, fourteen murine submissions with high
homology to human MNEI (SERPINB1) were identified using a
BLASTn search. Sequencing of selected clones revealed one full-length
clone (I.M.A.G.E. Consortium CloneID 63727). The thirteen
partial clones were 97-100% identical to the full-length clone,
strongly indicating that all are products of the same gene, which was
named EIA. However, Southern blot analysis of digested genomic DNA
using two EIA probes in exon 7 coding and non-coding regions revealed
three bands, indicating the presence of three or more homologous genes
in the mouse genome (Fig. 1).
Four genes with homology with SERPINB1 were fully sequenced
as follows. Mouse genomic DNA lambda clones containing EIA and a
homologous gene, EIB, were isolated after screening with a
32P-labeled EIA exon 7 probe and sequencing PCR products
with primers based on exon 7 sequence. The sequencing data obtained
from SacI subclones of lambda clone #1 containing EIA and
from BamHI subclones of lambda clone #3, containing EIB,
provided the sequence from exon 5 to 7 and several kilobases of
3'-flanking region of both genes. Using a PAC DNA library and primers
based on cDNA sequence of exons, the sequence for EIA and EIB
introns were obtained from PAC clones 507G8 and 672N8, respectively.
Amplification from exon 4 to 5 of EIA using low stringency conditions
produced two additional fragments with a small difference in size.
Sequencing identified these as fragments of two new genes, EIC and EID.
EIC gene was fully sequenced from exon 1 to 7 from PAC 507G8 using
primers based on cDNA sequences obtained from RT-PCR studies (see
below). EID putative exons were identified by searching the Celera data base with exon sequences of the other mouse EI genes. EID was sequenced
using primers designed on genomic DNA sequences of putative exons 2, 4, 5, and 6 from BLAST searches of the Celera data base using EIA and EIC
exon sequences. The structure of human MNEI and the four murine
homologs is shown in Fig. 2.
The complete sequencing of the four identified mouse genes showed both
conserved and different features compared with human MNEI gene,
SERPINB1 (Fig. 2). EIA and EIB had a similar seven-exon structure with exon size and codon phasing identical to those of human
MNEI. The introns sizes were also similar. EIC shared these features
and only differed in that an unusually large (~8.0kb) intron II was
present, when compared with other ov-serpin genes. The sequence
analysis of this intron revealed that this was due to the insertion of
a murine endogenous retrovirus-like (MuERV-L) element of ~6.5 kb
widely found in the murine genome (27, 28). In RT-PCR analysis (see
below), it appeared that this large intron includes an alternatively
spliced exon 2b, which contains a premature stop codon.
Although EID contains structural similarities with MNEI, no putative
exon 3 or exon 7 were found in the Celera data base nor in the
sequenced PAC 507G8 fragment amplified from exon 2 to 4. Moreover,
exons 2 and 5 contain several stop codons, and both exons induce a
codon phase shift, because they are one base pair longer or shorter
than the consensus, respectively. The base pair identity score of the
overlapping cDNA coding sequences for the four murine genes is
85-89% when compared with each other and 74-79% when compared with
MNEI, with EIA being the closest to MNEI.
mRNA Expression in Tissues and EIC Splicing Variants--
Due
to the high level of sequence identity between the four mouse EI genes,
the specificity of each reaction was confirmed in control PCRs with
cloned cDNA templates of EIA, EIB, and EIC and cloned genomic
template of EID. EIA mRNA was expressed in all tissues
investigated, and the signals were comparable to those of
To characterize the EIC PCR product, a second round of PCR was
performed with the same primer pair (exons 2 and 7). Four products were
sequenced, and all arose from EIC by alternative splicing (Fig.
3B). Two of these variants contained premature stop codons (indicated by asterisks in Fig. 3B). The two
other variants lacked the endogenous retrovirus exon 2b as well as
exons 4 and 5 or exon 5 and 64 bp of exon 6. If these messages are
translated into proteins, they are unlikely to be functional serpins
because they all lack exon 5, which encodes for strand 3 of
A further degree of complexity into EIC structure was added with the
sequencing results of 5'-RACE. Several fragments were cloned and
sequenced after two rounds of PCR using nested primers specific for
exon 3 of EIC. One product contained sequences of exons 1, 2, and 3 of
EIC but not exon 2b. Interestingly, another product contained exon 3 of
EIC preceded by 555 bp with high homology with another murine
ov-serpin, R86, found in the same locus on chromosome 13 (29). Analysis
of the Celera genomic data base revealed a gene remnant that mapped
~40 kb upstream of EIC and consisted only of exons 1 and 2; it was
named R86B. To rule out a cloning artifact during 5'-RACE, the
expression of the R86B/EIC chimeric mRNA was re-examined by RT-PCR
using a R86B-specific exon 2 forward primer together with the same
reverse primer in exon 7 of EIC. The expression level and tissue
pattern was identical to that of EIC (Fig. 3A). Furthermore,
the R86B/EIC band was also smaller than expected corresponding to a
splicing product unlikely to produce a functional serpin.
Formation of Stable Proteinase-Inhibitor Complexes--
The amino
acid sequence and the length of the RCL are critical in defining the
target range and the efficency of the inhibitory function of serpins.
Fig. 4 shows an alignment of EIA, EIB,
and EIC RCL with MNEI and other relevant human and murine serpins. EIA
and MNEI show a strikingly conserved RCL length and sequence with
identical putative P1 and P1' residues. It is predictable that MNEI and
EIA share protease targets, whereas EIB would target a different range
of enzymes. EIC, however, has two residues (Thr and Asp) in the hinge
region, which are likely to interfere with a correct insertion of the
RCL into The length and the sequence of the reactive center loop are
critical for the inhibitory activity of serpins (6, 7). MNEI and EIA
share the same Cys-Met residues at position P1-P1', and the lengths of
the RCL are identical. Complex formation assays confirmed that
EIA and MNEI share the same target proteases, because EIA formed stable
complexes with the three neutrophil granule proteases, elastase,
cathepsin G, and proteinase-3, and with pancreatic elastase and
chymotrypsin. Furthermore, MNEI and EIA are both expressed in a wide
range of tissues with the highest levels of expression in bone marrow,
spleen, and pancreas. Interestingly, these tissues contain cells that
constitutively produce large amounts of elastase and other target
proteases, indicating that the presence of these serpins may protect
against unwanted protease leakage, as previously proposed (14, 17). The
constitutive tissue expression of MNEI and EIA in many human and mouse
tissues indicates that these molecules may also limit the
tissue-damaging effects of neutrophil proteases in inflammatory
processes. This view is supported by the protective effects of
instilled recombinant human MNEI on elastase-induced pulmonary injury
in rats (21). This strongly supports the development of inhaled
recombinant MNEI as a potential aerosol treatment for inflammatory lung
diseases, including cystic fibrosis. However, although extracellular
MNEI was shown to be a strong inhibitor of neutrophil elastase, the mechanism of release of MNEI from the cytosol to the extracellular milieu remains to be demonstrated and may involve an alternative secretion pathway, because ov-serpins lack signal peptide for secretion. Furthermore, PI-6, a closely related serpin, failed to be
secreted from activated and unactivated platelets and various cultured
cell lines (30). It was also shown that overexpression and the addition
of an N-terminal signal peptide did not result in secretion of the
protein. Therefore, it is also possible that the ubiquitous expression
of MNEI in humans and EIA in mouse is required for the inhibition of
other proteases with a wider expression pattern than neutrophil
proteases or pancreatic elastase. We recently showed that MNEI
inhibits mast cell chymase, chymotrypsin, and prostate-specific antigen
but not granzymes, trypsin-like serine proteases, or cysteine and
metalloprotease family members (31). The role of MNEI and EIA at
inhibiting proteases with chymotrypsin-like specificity in
vivo may provide further understanding of the conserved wide
tissue range of expression in these species.
MNEI is the product of a single gene (SERPINB1), which is
located on chromosome 6p25 (32) together with two other
seven-exon ov-serpins, PI-6 (SERPINB6) and PI-9
(SERPINB9) (33). In a recent mapping study, we have shown
that the four murine MNEI homologs are located on chromosome 13 together with PI-6- and PI-9-related genes, in a region syntenic
to human 6p25 (29). The analysis of the mouse locus shows that it is
likely that the common ancestor locus contained three genes
corresponding to MNEI, PI-6, and PI-9, and that the murine gene
repertoire expansion occurred after the two species diverged (29). This
hypothesis is supported by the presence of several pseudogenes and
remnants in the mouse locus, whereas none have been identified in the
human locus. If EIA is functionally similar to MNEI, what is the role
of the three other paralogs? The gene and predicted protein structures
of EIB indicate that it is a biologically active serpin. This was
confirmed by its ability to form stable inhibitory complexes with
cathepsin G, a protease with chymotrypsin-like specificity. However,
EIB failed to inhibit elastase-like proteases, which was predicted by
the great divergence of its RCL. Moreover, EIB is proteolytically degraded by elastases, proteinase-3, and chymotrypsin. EIB also differs
from MNEI in that it has a lower level and restricted tissue range of
mRNA expression. This demonstrates that EIB does not duplicate the
activity of EIA.
The sequencing data show that EIC has the same gene structure as EIA
and EIB with identical exon sizes and similar intron size, except that
the insertion of a full-length MuERV-L element into what once was
intron II has modified this region of the gene leading to the
transcription of the alternatively spliced exon 2b (Figs. 2 and
3B). The insertion of such sequences within genes has been
shown to induce alternative splicing events involving the long terminal
repeat (LTR) region of the endogenous retrovirus (34-36). In contrast,
exon 2b of EIC corresponds to the antisense strand of the protease
region of the pol gene. The pol gene,
unlike the LTR, has no polyadenylation signal and therefore allows the transcription to carry on to the downstream exons of EIC. The mechanisms that produce the atypical splicing events involving exons 4, 5, and 6 of EIC as well as the intergenic variants of R86B/EIC are
unclear because the exon/intron junctions are conserved. In any case,
the cDNA sequences show that it is unlikely that any of the EIC
splicing variants would translate into a biologically active serpin.
Furthermore, the presence of polar and charged residues in the proximal
hinge region of the RCL are unfavorable for serpin inhibitory activity.
The second and fourth splicing variants described in Fig. 3B
may produce proteins with yet unknown functions. Evolutionarily
interesting splicing variants have been described in insect serpins
where the exon containing the RCL is present in twelve alternative
forms, which are mutually exclusive, offering a powerful way of
expanding the target repertoire (37, 38). Alternative splicing was also
observed in human ov-serpins, squamous cell carcinoma antigen (SCCA) 1 and 2 (SERPINB3 and 4) and hurpin
(SERPINB13), but their biological relevance remains unclear
(39, 40). Similarly to EIC, mRNA splicing variants containing the
first two exons of R86B and the downstream exons of EIC are unlikely to
be functional serpins. However, the possibility that such chimeric
proteins may be produced indicates that the repertoire of serpins may
be even larger than that predicted by the number of genes.
EID lacks important features of an active serpin at the genomic level,
and no mRNA transcript has been detected in any tissue: it is
clearly a pseudogene. The absence of exon 7 in EID resolves the
discrepancy between the Southern blot analysis and the number of genes,
because the screening was done using exon 7 probes (Fig. 1). Moreover,
the restriction map of the fully sequenced mouse genes (not shown) is
consistent with the size of the bands observed in Fig. 1, indicating
that it is unlikely that another gene with homology with MNEI and with
a functional RCL (encoded in exon 7) is present in the mouse genome.
The four murine ov-serpins described here are homologous to human MNEI
(SERPINB1) but have met different evolutionary fates. According to the new serpin nomenclature guidelines (2), the genes
encoding EIA, EIB, and EIC will be called Serpinb1,
Serpinb1b, and Serpinb1c, respectively. Because
it is a pseudogene, EID will be called Serpinb1-ps1.
Similarly, R86B is a pseudogene showing sequence homology with PI-9 and
will therefore be called Serpinb9-ps2. We can conclude that,
based on mRNA expression pattern and protease targets, EIA is the
functional murine counterpart of MNEI and that, based on genomic
mapping and sequence homology, it is likely that EIA is the mouse
ortholog of MNEI. The phenotype of mice lacking EIA
(Serpinb1) will provide interesting insight on the role of
MNEI in regulating the activity of granule proteases against pathogens
and in inflammatory diseases.
We are grateful to Dr. Michael Carroll and
Dr. Judy Lieberman for materials.
*
This work was supported by the National Institutes of Health
Grant HL66548-02 and a Pilot Grant from the Cystic Fibrosis Foundation.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 GenBankTM/EBI Data Bank with accession number(s) AF521697, AF521698, AF521699, and AF521700.
¶
To whom correspondence should be addressed: The Center for
Blood Research, 800 Huntington Ave., Boston MA 02115. Tel.:
617-278-3314; Fax: 617-278-6613; E-mail:
benarafa@cbr.med.harvard.edu.
Published, JBC Papers in Press, August 19, 2002, DOI 10.1074/jbc.M207080200
The abbreviations used are:
RCL, reactive center
loop;
CatG, cathepsin G;
Chy, chymotrypsin;
GrzB, granzyme B;
LTR, long
terminal repeat;
MNEI, monocyte neutrophil elastase inhibitor;
MuERV-L, murine endogenous retrovirus-like;
PR3, proteinase-3;
PI, proteinase
inhibitor;
SCCA, squamous cell carcinoma antigen;
RACE, rapid
amplification of cDNA ends;
hNE, human neutrophil elastase;
pPE, porcine pancreatic elastase;
RT, reverse transcriptase;
Bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol;
MES, 4-morpholineethanesulfonic acid;
PAC, P1 phage artificial
chromosome.
Characterization of Four Murine Homologs of the Human ov-serpin
Monocyte Neutrophil Elastase Inhibitor MNEI (SERPINB1)*
§¶,,,
, and§
Center for Blood Research and
§ Department of Pediatrics, Harvard Medical School, Boston,
Massachusetts 02115 and the Department of Biochemistry and
Molecular Biology, Monash University,
Victoria 3800, Australia
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-sheet A of the molecule, which results in the pole to pole
displacement of the protease (4, 5). The recent, and long awaited,
crystal structure of a covalent serpin-protease inhibitory complex has
shed light on the mechanism of inhibition of serpins (6). The full
insertion of the RCL results in a hyperstable serpin molecule and leads
to the distortion of the protease by pulling away the reactive serine
from its catalytic partners. This mechanism of inhibition by distortion
is dependent on the length of the RCL (6, 7).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-actin were also assayed using primers
described previously (24).
Sequence of gene-specific primers used in RT-PCR
-actin are 725, 529, 972, 1,035, 493, and 537 bp, respectively.
20 °C.
Purified recombinant murine granzyme B (mGrzB) (26) was a generous gift
from Dr. Judy Lieberman (Center for Blood Research, Boston, MA) and was
kept in 0.5 mg/ml aliquots at 20 °C.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Determination of gene copy number by Southern
blot analysis. Mouse genomic DNA was digested with the indicated
restriction enzymes and hybridized as indicated with an exon 7 coding
region (CR) or a 3' untranslated region
(3'UTR) probe. Both probes hybridized with three
fragments generated by all restriction endonucleases. Note that one
band generated with the 3'-UTR probe and EcoRI is a doublet.
DNA size markers (kb) are shown on each side.
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Fig. 2.
Gene structure and organization of the mouse
homologs of MNEI. Exons, which are drawn to scale, are represented
as black (coding regions) and gray (non-coding
regions except the first 8 bp of exon 2) boxes.
Numbers in boxes indicate exon size in base pairs
(bp). Introns, which are not drawn to scale, are shown as
lines linking exons and their sizes are shown in base pairs.
Note that the seven-exon ov-serpin structure is conserved. The start
codon is at position 9 in exon 2 of all genes, and the reactive center
as well as the stop codon are in exon 7. Also note that the coding
exons 2 to 6 of EIA, EIB, and EIC are identical in size to MNEI and
that the variability in exon 7 is mainly due to the length of the
3'-untranslated region. EIC contains a 178-bp alternatively spliced
exon 2b, encoded by a murine endogenous retrovirus-like (MuERV-L)
sequence. The region between exon 2 and 3 of EIC has been detailed to
show the reverse orientation and the position of the ~6.5-kb MuERV-L
sequence. Exon 2b is encoded on the antisense strand of the MuERV-L
pol gene. EID lacks exons 3 and 7. The official gene
nomenclature and the GenBankTM accession numbers are shown
on the left.
-actin,
except in bone marrow, spleen, and pancreas, where EIA mRNA
expression was higher than that of -actin, and in heart and liver
where a lower expression level was observed (Fig.
3A). EIB signal was comparable
to that of EIA in brain, but the signal was one to two orders of
magnitude lower in lung, spleen, and testis, and no amplification was
seen in the other tissues. Interestingly, EIC was only detectable in
heart, but the product (~830 bp) was smaller than expected (972 bp,
from exon 2 to 7). EID could not be amplified from any tissue.
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Fig. 3.
Expression profiles of mouse EI genes.
A, tissue-specific mRNA expression of mouse
EI genes. Mouse tissue RNA were amplified by RT-PCR using EIA-, EIB-,
EIC-, and EID-specific primers. -Actin was amplified as a
ubiquitously expressed mRNA control. Note that EIA is expressed at
high levels in all tissues, especially in bone marrow, spleen, and
pancreas. EIB is mainly expressed in brain. EIC and the chimeric
transcript R86B/EIC (see text) are only expressed in heart. Both
products are smaller than those predicted by the gene organization (see
text). EID was not detected in any tissues (not shown). B,
splicing variants of EIC expressed in heart. The asterisks
indicate the presence of premature stop codons. Note that all splicing
variants lack exon 5 either alone or in combination with exon 4 or with
64 bp of exon 6, which contains an internal cryptic splice site. The
first three variants also lack exon 2b, which is encoded in the MuERV-L
sequence.
-sheet
A, a critical structure for the correct insertion of the RCL.
-sheet A. To verify their predicted function, EIA and EIB
were produced in vitro with 35S-label and
allowed to form stable covalent complexes with several proteases (Fig.
5). EIC and EID were not tested because
no full-length EIC cDNA could be identified and EID lacks exon 7, which contains the RCL. EIA formed SDS-stable complexes with human
neutrophil elastase, porcine pancreatic elastase, human cathepsin G,
bovine chymotrypsin, and human proteinase-3 but not with granzyme B
(Fig. 5A). When tested in parallel with the same proteases,
EIB could only form a stable complex with cathepsin G but at a lesser
efficiency than EIA (not shown). To make the case clearer, EIB was
incubated for up to 30 min with hCatG or hNE, and the total amount of
protein electrophoretically separated was doubled (Fig.
5B).
View larger version (33K):
[in a new window]
Fig. 4.
Reactive center loop sequences suggest
different protease targets for mouse homologs of MNEI. Amino
acid sequences of reactive center loops of EIA, EIB, and EIC
were aligned manually with those of human MNEI, proteinase inhibitor
(PI)6, PI9, squamous cell carcinoma antigen 2 (SCCA2) and
1-antichymotrypsin (AACT). EIA and MNEI have almost identical
sequences in this region underscoring possible similar protease
targeting. Target proteases for human serpins are indicated on the
right. Hydrophobic and apolar residues are indicated in
yellow, acidic residues are in red, basic
residues are in blue, and polar residues are in
green. The asterisk indicates the position of the
principal P1 residue for these serpins.
View larger version (81K):
[in a new window]
Fig. 5.
EIA and EIB form stable inhibitory complexes
with different sets of serine proteases. A,
35S-labeled EIA, generated by in vitro
transcription/translation, was incubated (in 20 µl) at 37 °C for 5 min with the indicated amounts of neutrophil elastase (hNE),
pancreatic elastase (pPE), cathepsin G (hCatG),
chymotrypsin (bChy), proteinase-3 (hPR3), and
granzyme B (mGrzB). Phosphorimages are shown. Native EIA is
indicated by filled arrowheads to the left of
each panel. Inhibitory complexes (cpx) are
indicated by open arrowheads on the right of
each panel. Note that EIA forms inhibitory complexes with
hNE, pPE, hCatG, bChy, and hPR3. In these conditions, EIB forms
complexes with hCatG only and is proteolytically degraded by hNE, pPE,
bChy, hPR3, but not mGrzB (not shown). B,
35S-labeled EIB was incubated at 37 °C for the indicated
time with 200 ng of hCatG or hNE. Note that EIB forms stable complexes
with hCatG, whereas it is degraded by increasing concentrations of hNE
without formation of complex, indicating that EIB is a substrate for
this protease.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ACKNOWLEDGEMENTS
FOOTNOTES
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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