SPAI-2 (
)is a 61-amino-acid peptide isolated from
porcine duodenum as a Na
,K
-ATPase
inhibitor (1) and is now considered as an inhibitor specific
for monovalent cation-transporting ATPases(2) ; however,
whether this inhibitory action observed in vitro is its true
physiological function remains to be established since the IC
is relatively high (
10 µM). It belongs to the
WAP protein superfamily that has the ``four-disulfide core'' (3) or ``WAP'' motif(4) ; the members
include, among others, mucous proteinase inhibitor
MPI(5, 6, 7) and elafin, an elastase
inhibitor that was isolated by Wiedow et al.(8) from
the horny layers of patients with psoriasis. MPI and elafin are well
characterized at the molecular level; for example, their domain
structures and gene structures have been
determined(9, 10) . Less is known about the molecular
biology of SPAI. We therefore cloned cDNA from a porcine duodenum cDNA
library, determined its nucleotide sequence to deduce the primary
structure of the precursor, and examined the exon-intron organization
of the corresponding gene. The results indicated that SPAI is much more
similar to elafin than to MPI in its domain and gene structures,
suggesting that SPAI and elafin are, despite their difference in
biological activities, evolutionarily very close among the members of
the WAP superfamily.
At present, the physiological significance of
SPAI is not clear. Localization often offers valuable clues to the
physiological roles. We therefore also examined 1) tissue distribution
of the SPAI message by Northern analysis and 2) whether SPAI is present
in plasma by immunoaffinity chromatography.
EXPERIMENTAL PROCEDURES
Materials
Fresh porcine tissues were obtained
from the Shibaura abattoir sanitation inspection station, Tokyo, Japan.
Restriction enzymes were obtained from Toyobo, Osaka, Japan; random
primer DNA labeling kit version 2 was from Takara, Kyoto, Japan;
Sequenase version 2.0 sequencing kit was from U. S. Biochemical Corp;
P-labeled nucleotides were from DuPont NEN. Rabbit
polyclonal antiserum to porcine SPAI was kindly donated by Peptide
Institute Inc., Osaka, Japan. Monoclonal antibodies against porcine
SPAI were obtained by standard hybridoma techniques. The hybridoma
producing the monoclonal antibodies 1F4 and 1H12 were amplified in the
ascites fluid of mice. The IgG produced by the ascites induction was
then purified on Protein A-Sepharose 4 FF (Pharmacia Biotech Inc.)
according to the instructions of the manufacturer. Monospecificities of
the antibodies were established by Western blotting; they stained only
the SPAI band among various protein bands when proSPAI, prepared as
described below, was mixed with pig kidney extracts and analyzed by
SDS-PAGE and immunostaining(11) .
Preparation of the Oligonucleotide Probe
SP-P5A
The oligonucleotide used for cDNA library screening was
designed according to the nucleotide sequence of short PCR products.
The following PCR also served as a test for the presence of SPAI
message. Poly(A)
RNA (170 µg) was isolated from
porcine duodenum (2 g) using a FastTrack mRNA isolation kit. A mixture
of oligo(dT)-primed cDNA was synthesized from the duodenal mRNA using
reverse transcriptase and used as templates for PCR. The primers used
for PCR were made based on the consensus amino acid sequence of the
three SPAIs reported previously(1) ; the consensus regions
selected were those corresponding to amino acid residues 34-40
(5` primers) and amino acid residues 52-57 (3` primers) of
SPAI-2. The sequences of the 5` primers were:
CAAGCTTGAA(T/C)AA(A/G)TG(T/C)TGGCG(T/C)GA(T/C)TA (primer 51),
CAAGCTTGAA(T/C)AA(A/G)TG(T/C)TGGCG(A/G)GA(T/C)TA (primer 52), and
CAAGCTTGAA(T/C)AA(A/G)TG(T/C)TGGAG(A/G)GA(T/C)TA (primer 53); the
sequences of the 3` primers were:
GGAATTCCCA(A/G)TC(T/C)TT(A/G)CC(A/G)CA(A/G)AA (primer 31) and
GGAATTCCCA(A/G)TC(T/C)TT(T/C)CC(A/G)CA(A/G)AA (primer 32). The 5` ends
of the 5` and 3` primers contained the HindIII and EcoRI restriction sequences, respectively, for the subsequent
cleavage of cloned cDNA fragments. All six possible combinations of the
primers (three 5` primers versus two 3` primers) were examined
using a GeneAmp DNA amplification kit (Perkin-Elmer) according to the
following schedule: 94 °C for 1 min, 44 °C for 2 min, and 72
°C for 3 min for 30 cycles, followed by further incubation for 7
min at 72 °C. Only two combinations (primer 53 versus primer 31 and primer 53 versus primer 32) yielded
amplified products of about 80 bp. After electrophoretic purification
on an agarose gel, the products were treated with T4 polymerase and T4
kinase, subcloned into M13 mp18 at the SmaI site and
sequenced. The sequence flanked by the primers were
5`-TGACTGTCCAGGGGTCAAGAAGTGCTGTGAAGGC-3` (SP-P5) and corresponded, as
expected, to amino acid residues 40-51 of SPAI-2; therefore, the
antisense sequence (SP-P5A) complementary to SP-P5 was chemically
synthesized and used as an oligonucleotide probe for screening.
Construction and Screening of cDNA Library
A cDNA
library was constructed, using an Amersham cDNA cloning system, in
gt10 from porcine duodenum poly(A)
RNA isolated
with a FastTrack mRNA isolation kit (Invitrogen). Plaques were screened
using the Escherichia coli strain NM514 by hybridization of
P-labeled SP-P5A, a synthetic 36-base antisense probe, to
nitrocellulose replicates in 50 mM sodium phosphate buffer, pH
7.0, containing 60 mM sodium citrate, 0.6 M NaCl, 1
Denhardt's (1
Denhardt's: 0.1% each of
bovine serum albumin, polyvinylpyrrolidone, and Ficoll) at 65 °C.
Filters were washed in a solution containing 0.6 M NaCl, 60
mM sodium citrate, and 50 mM sodium phosphate (pH
7.0) at 65 °C. Positive clones were plaque-purified, and the cDNA
inserts were subcloned into pBluescript II (Stratagene) for sequencing.
Screening of Porcine Genomic Library
A porcine
liver genomic library, constructed in
EMBL3 SP6/T7 (Clontech), was
used. Approximately 300,000 plaques were screened on nitrocellulose
filters by hybridization to the 700-bp SPAI-2 cDNA insert labeled with
dCTP by random primer labeling.
Hybridization was carried out for 16 h at 37 °C in 50% formamide plus 5
SSPE (1
SSPE: 0.15 M NaCl, 10
mM sodium phosphate, pH 7.4, and 1 mM EDTA), 5
Denhardt's, and 0.1% SDS. The filters were rinsed twice
at room temperature in 2
SSC (1
SSC: 0.15 M NaCl, 15 mM sodium citrate, pH 7.0) containing 0.05% SDS
and twice at 55 °C in 1
SSC containing 0.1% SDS for 1 h.
Autoradiography was for 24 h at -70 °C with Kodak X-Omat AR x-ray
film (Eastman Kodak Co.).
Southern Blot Analysis of Porcine Genomic
DNA
Porcine genomic DNA was isolated from liver. Ten µg of
the DNA were digested with EcoRI, HindIII, and XbaI. The digests were electrophoresed in a 0.7% agarose gel
at 0.6 V/cm and transferred to Magnagraph nylon membrane (Micron
Separations Inc.) using an LKB VacuGene vacuum blotting unit. The
membrane was prehybridized at 42 °C for 2 h and hybridized with the
P-labeled XbaI-PvuII fragment (561 bp)
derived from SPAI-2 cDNA at 42 °C in 50% formamide, 5
SSPE,
5
Denhardt's, and 0.1% SDS and followed by washes and
autoradiography as described above.
Northern Hybridization
Total RNA was prepared from
the porcine tissues described in Fig. 6using either the
guanidinium thiocyanate-CsCl method (12) or acid guanidinium
thiocyanate-phenol-chloroform extraction method(13) . Twenty
µg of total RNA were denatured in 50% formamide, 16% formaldehyde,
20 mM Mops, pH 7.0, 5 mM sodium acetate, and 1 mM EDTA, for 15 min at 65 °C, electrophoresed in a 1.0% agarose
gel containing 2.2 M formaldehyde at 3 V/cm, and transferred
to nylon membrane. The membrane was hybridized with
P-labeled SPAI-2 cDNA probe (XbaI-PvuII
fragment) at 42 °C in 50% formamide, 5
SSPE, 5
Denhardt's, and 0.1% SDS and washed twice at room temperature
with 2
SSC containing 0.05% SDS, twice with 1
SSC
containing 0.1% SDS at 55 °C for 2 h, and finally with 0.5
SSC containing 0.1% SDS at 55 °C for 8 h.
Figure 6:
Intestine-specific expression of SPAI mRNA
revealed by Northern blot analysis. About 20 µg of total cellular
RNA isolated from the indicated tissues was fractionated on a
denaturing gel and transferred to a nylon membrane filter. The filter
was probed with a radiolabeled cDNA probe and autoradiographed. Equal
loading of RNA is demonstrated by the ethidium bromide staining of the
28 and 18 S rRNA bands (lower
panel).
Enzyme-linked Immunosorbent Assay
To determine
plasma SPAI levels and to monitor purification of SPAI-LI, the
following sandwich ELISA was devised, consisting of solid-phase
monoclonal antibody 1H12 as the capture reagent and biotinylated
monoclonal antibody 1F4 as the second antibody. Microtiter plates were
coated with affinity-purified monoclonal antibody 1H12 (10 µg/ml)
in 20 mM phosphate-buffered saline, pH 7.4, at 4 °C
overnight. After blocking with Block Ace (Dainippon Seiyaku; diluted
4-fold in H
O) for 1 h at room temperature, the wells of the
plates were loaded with 100 µl of samples and allowed to stand for
1 h at room temperature followed by washing with 0.9% NaCl containing
0.05% Tween (saline-Tween). Subsequently the wells were incubated at
room temperature for 1 h with a 10-fold diluted Block Ace solution
containing biotinylated monoclonal antibody 1F4 (4.5 µg/ml), which
had been prepared by coupling the purified 1F4 IgG with N-hydroxysuccinimide biotin (Pierce). Following washing with
saline-Tween, to quantitate the sandwiched antigen, the wells were then
incubated for 2 h at room temperature with 100 µl of horseradish
peroxidase avidin D (Vector Laboratories) (0.2 µg/ml of Block Ace
diluted 1:10 with water), and developed with o-phenylenediamine (Sigma) and hydrogen peroxide as
substrates.
Purification of SPAI-like Immunoreactivity from
Plasma
Porcine serum (1 liter) was acidified to pH 2.0 with 2 N HCl and filtered through no. 2 qualitative filter papers
(Advantec) to remove precipitates. The filtrate was loaded onto a
silica column (Sep-Pak Vac, Waters), which had been equilibrated with
0.1% trifluoroacetic acid. After washing with 0.1% trifluoroacetic
acid, the column was eluted with 15% CH
CN containing 0.1%
trifluoroacetic acid. The eluate was lyophilized, redissolved in 75 ml
of 20 mM Tris-HCl buffer, pH 7.4, containing 0.5 M NaCl, and applied to an immunoaffinity column (1.5
3 cm),
which had been prepared by cross-linking 5 mg of the anti-SPAI
monoclonal antibody 1H12 to 5 ml of CNBr-activated Sepharose 4B
(Pharmacia) according to the manufacturer's instruction, at a
flow rate of 5 ml/h and equilibrated with 20 mM Tris-HCl, pH
7.4, containing 0.5 M NaCl. After washing the column with the
same buffer, the absorbed proteins were eluted with 0.1 M Gly-HCl, pH 2.5, lyophilized, and dissolved in 1 ml of 10%
CH
CN containing 0.1% trifluoroacetic acid. The
affinity-purified sample was further purified by reverse-phase HPLC on
a YMC-GEL C
column (4.6
250 mm). The HPLC column
was eluted first by an isocratic mode with 25% CH
CN
containing 0.1% trifluoroacetic acid for 3 min and then by a linear
gradient from 25% to 30% CH
CN containing 0.1%
trifluoroacetic acid for 40 min at a flow rate of 1 ml/min monitoring
absorbance at 225 nm. Every peak was collected and assayed for porcine
SPAI-LI.
Sequencing of Plasma SPAI-LI
The SPAI-LI purified
by immunoaffinity chromatography and HPLC was sequenced after
carboxymethylation, fragmentation with chymotrypsin, and, in the case
of the N-terminal fragment, pyroglutamyl peptidase treatment.
SPAI-LI
(2 nmol), the major peak obtained by the C
HPLC, was dissolved in 200 µl of 1 N Tris-HCl, pH
8.6, containing 3.5 M guanidine and 0.25% EDTA and reduced
with 20 µl of 0.6 M dithiothreitol under N
gas. Following a 2.5-h incubation at 37 °C, 30 µl of 0.6 M carboxymethyliodide and 30 µl of 0.5 N NaOH
were added to the mixture. Carboxymethylated SPAI-LI
was
purified by HPLC on an ODS column (4.6
250 mm, YMC) using a
30-min acetonitrile gradient from 26% to 34% containing 0.1%
trifluoroacetic acid at a flow rate of 1 ml/min. The carboxymethylated
SPAI-LI
preparation was then dissolved in 200 µl of 0.1 M Tris-HCl, pH 8.0, and digested with 1 µg of N
-p-tosyl-L-lysine
chloromethyl ketone-treated chymotrypsin (Sigma) at 37 °C for 2.5
h. The digest was chromatographed on the 4.6
250-mm ODS column,
and five peaks were obtained using a 60-min 1-32% gradient of
acetonitrile containing 0.1% trifluoroacetic acid. The retention times
of the first four peaks coincided with those of the chymotryptic
fragments of the carboxymethylated form of the mature SPAI previously
isolated from porcine duodenum(1) , and their identities were
confirmed by sequencing. The fifth peak, which was considered to be the
N-terminal fragment, was lyophilized, redissolved in 100 µl of 0.1 M Tris-HCl, pH 8.0, and treated with 755 milliunits of
pyroglutamate aminopeptidase (Nakarai) at 37 °C for 6 h. The
reaction was monitored by the reverse phase HPLC described above using
a 40-min acetonitrile gradient from 25 to 30%. The cleaved peptide was
isolated with the same HPLC condition and sequenced in an Applied
Biosystems 473A pulsed liquid sequenator.
Construction of MBP-proSPAI Expression Vector
A
cDNA fragment coding for porcine proSPAI was prepared by PCR using SPAI
cDNA as template. The following oligonucleotides were synthesized to
amplify a segment of the porcine SPAI cDNA sequence corresponding to
amino acid residues 21-167 (Fig. 2): The sense primer was
a 17-mer (5`-GCGCAGAGACTTGACAG-3`), and the antisense primer was a
24-mer (5`-GAAGCTTTCACTTGGGATACAAAC-3`). The PCR reactions were carried
out with Taq DNA polymerase for 30 cycles (1 min at 94 °C,
0.4 min at 47 °C, and 2.5 min at 75 °C). The resulting PCR
product was purified, blunt-ended, digested with HindIII, and
ligated into the StuI/HindIII site of the pMAL-p
vector (New England Biolabs). The ligations were used to transform E. coli XL1-Blue, and the desired recombinants were selected
by sequencing their plasmid DNA (pMAL-p-proSPAI).
Figure 2:
Complete amino acid sequence of porcine
SPAI predicted from cDNA and genomic DNA clones. Partial nucleotide
sequences of the two introns and their locations are shown by downward arrows. cDNA clone 57G covers the sequence from
nucleotide -6 (rightward arrow) to nucleotide 722 (leftward arrow). Slash between nucleotides 722 and
723 indicates the poly(A) tail found in cDNA. Signal sequence and
prosequence cleavage sites are indicated by arrowhead and upward arrow, respectively. Note that the
``prosequence'' cleavage site
(Asp
-Pro
) is acid-labile and is readily
cleaved when exposed to strong acid(15, 16) . Eight
Cys residues involved in the formation of four-disulfide core are shaded.
Expression and Purification of ProSPAI
Production
of MBP-proSPAI and cleavage of the fusion protein with Factor Xa were
carried out using a New England Biolabs expression kit exactly as
described by the manufacturer. Briefly, XL1-Blue cells transformed with
pMAL-p-proSPAI were grown at 37 °C for 4 h and the expression of
the MBP-proSPAI fusion protein was induced by adding
isopropylthiogalactoside. Following a 2-h incubation, the cells were
pelleted, washed with 20% sucrose, and treated with 5 mM MgCl
on ice, and the periplasmic fraction (cold
osmotic shock fluid) was recovered by centrifugation. Cold osmotic
shock fluid (1.5 l) was applied to a column containing amylose resin
(100 ml), and the MBP-proSPAI fusion protein was eluted with 10 mM maltose. The eluate, containing 70 mg of protein in 40 ml, was
dialyzed against 50 mM Tris-HCl, pH 8.0, containing 100 mM NaCl and 1 mM CaCl
, cleaved with Factor Xa
(10 µg; Danex Biotek, Mundelstrup, Denmark) at 20 °C for 10 h,
and dialyzed against 10 mM phosphate, pH 7.2. The final
purification of proSPAI was achieved by removing the MBP fragment by
HPLC on a hydroxyapatite column (1.2
5 cm) equilibrated with 10
mM phosphate buffer, pH 7.2. When stained with Coomassie
Brilliant Blue after SDS-PAGE, proSPAI gave a reddish band that is
quite different from those of ordinary proteins that stain blue.
Other Procedures
Cross-linking of proSPAI with
transglutaminase(11) , SDS-PAGE(14) , production of an
anti-proSPAI antiserum(11) , and immunoblotting (11) were all performed according to the published methods.
RESULTS
Isolation and Sequence Analysis of Porcine SPAI
cDNA
A
gt10 cDNA library was prepared from porcine duodenum
and screened with an oligonucleotide probe, SP-P5A, designed from the
amino acid sequence of SPAI-2(1) . This screening (of about 3
10
recombinants) yielded one positive clone. The
positive phage clone was named
57G and characterized. The insert
was excised with EcoRI and subcloned into the EcoRI
site of pBluescript II for sequencing. A restriction map and sequencing
strategy for the cDNA clone are shown in Fig. 1. Fig. 2shows the nucleotide and deduced amino acid sequence of
clone 57G. The cDNA insert consisted of a total of 741 bases, with 6
bases upstream of the ATG codon and a poly(A) tail. An open reading
frame of 561 bases encoded a protein of 187 amino acids. The protein
sequence contained a hydrophobic signal sequence at its N terminus, a
prosequence, and the SPAI sequence at the C terminus (Fig. 4C). One of the most striking features is the
presence of 16 repeats of a hexapeptide with the following consensus
sequence: GQDPVK, which is repeated 4 times intact and 12 times in part (Fig. 3). Since the Asp
-Pro
bond
that is assigned as the prosequence cleavage site is known to be
susceptible to strong acid hydrolysis and such acidic conditions were
used in our previous isolation of SPAI, it is highly likely that the
cleavage of Asp
-Pro
bond might be an
experimental artifact; however, for simplicity, we use, in this paper,
the term ``proSPAI'' for the higher molecular weight native
form of SPAI to distinguish it from the short form of 61 amino acids
that was isolated from acidified porcine duodenum(1) .
Figure 1:
Restriction map and sequencing strategy
of a porcine SPAI cDNA clone. The proposed coding region is boxed. The shaded box indicates the coding region for
mature SPAI-2.
Figure 4:
Restriction map (A) and
exon-intron organization (B) of the porcine SPAI gene and
structural features of cDNA (C). PanelA,
the 5.4-kb EcoRI fragment commonly found in positive genomic
clones is shown together with sites for typical restriction enzymes: E, EcoRI; B, BglII; H, HindIII; Sp, SpeI; S, SacI; X, XbaI. PanelB, a
scale drawing of the overall organization of the SPAI gene; the exons
are numbered from the 5` end. C, features of
mRNA/cDNA; the translation initiation codon ATG, stop codon TGA, and
poly(A) addition signal AATAAA are indicated by bold
letters.
Figure 3:
Alignment of 16 hexapeptide repeats that
constitute the prosegment of the SPAI precursor. Repetitive sequences
rich in Gln or Lys or both have been found in several transglutaminase
substrates: For example, human elafin contains 5 hexapeptide
repeats(10) ; the guinea pig seminal vesicle clotting protein
SVP-1, 8 repeats of 24 amino acids(34) ; human involucrin, 39
repeats of a decapeptide(35) ; rabbit cornifin, 13 repeats of
an octapeptide; and sheep trichohyalin, 25 full- or partial length
repeats of a 23-amino acid
sequence(36) .
Isolation and Characterization of the SPAI Gene
A
porcine genomic DNA library in
EMBL3 SP6/T7 was screened with
P-labeled full-length cDNA, and numerous phage clones were
obtained. One set of the positive clones contained a 5.4-kb EcoRI fragment that hybridized strongly to the probe. The EcoRI fragment was subcloned into the plasmid vector
pBluescript II for further characterization. After its mapping using
various restriction enzymes and Southern blot analysis of the resulting
fragments, SacI digestion was found to produce three fragments
appropriate for sequencing the exon-intron boundaries: a 1.3-kb
fragment with two XbaI sites one of which corresponds to the XbaI site in the cDNA sequence, a 1.2-kb fragment with a
unique SpeI site corresponding to that in the cDNA sequence,
and a 2.9-kb fragment. Sequence analysis of these fragments according
to the strategy shown in Fig. 4A indicated that the
porcine SPAI gene spans approximately 1.8 kb and is divided into 3
exons and 2 introns (Fig. 4B); detailed positions of
the introns are shown in the Fig. 2sequence. Fig. 5shows the result of Southern blot analysis performed to
determine the copy number of the porcine SPAI gene. Genomic DNA was
digested with three restriction enzymes, transferred to a filter, and
hybridized to the XbaI-PvuII fragment of SPAI cDNA.
In all digests, single dense bands and two or three faint bands were
detected under moderate stringency conditions (Fig. 5A); the faint bands disappeared under stringent
conditions (Fig. 5B). The sizes of the dense bands are
in good agreement with those predicted from the genomic restriction map
illustrated in Fig. 4A, indicating that the SPAI gene
is present as a single copy in the porcine genome. Three forms of SPAI
have been isolated(1) : SPAI-1, SPAI-2, and SPAI-3. SPAI-1 is a
N-terminally truncated form of SPAI-2. SPAI-2 and SPAI-3 are of the
same length and differ only at two positions (Arg
Gly, Ser
Gly); each substitution can be produced
by a single nucleotide change in the corresponding codon. The
substitutions are therefore probably allelic variants in the SPAI-2
gene. The additional bands with relatively weak hybridization signals
suggest the existence of at least two other closely related genes.
Figure 5:
Southern blot analysis of porcine genomic
DNA probed with SPAI cDNA. High molecular weight genomic DNA was
isolated from porcine liver, digested to completion with the indicated
restriction enzymes, electrophoresed on an agarose gel, transferred to
nylon membrane, and hybridized to the
P-labeled cDNA clone
57G. The filter was washed in 1
SSC containing 0.1% SDS at 55
°C for 2 h (A), followed by a 2-h wash at 65 °C (B), before autoradiography.
Tissue Distribution of mRNA
To define tissue
distribution patterns of SPAI, expression of the SPAI mRNA in various
porcine tissues was studied by Northern blot analysis. Total RNA
preparations from the brain, liver, lung, adrenal, stomach, small
intestine, large intestine, and kidney were electrophoresed on an
agarose gel, transferred to nylon membrane, and probed with
P-labeled cDNA. A transcript of about 900 nucleotides was
detected in the intestine but in none of the other tissues examined (Fig. 6). Within the intestine, SPAI mRNA was much more abundant
in small intestine than in large intestine.
A High Molecular Weight Form of SPAI in the
Circulation
We further examined SPAI levels in plasma by
immunoaffinity chromatography. A monoclonal antibody to SPAI, 1H12, was
produced, purified with Protein A, and coupled to Sepharose 4B.
Immunoreactive SPAI in porcine plasma was adsorbed to the
immunoaffinity gel, eluted with glycine-HCl, and further purified by
reverse-phase HPLC on a C
column (Fig. 7A). Chemical sequencing of the major peak
indicated that porcine plasma contains a SPAI species much larger than
the SPAI originally isolated from duodenum by us(1) . As shown
in Fig. 7B, the N terminus of the circulating form
corresponded to Gln at position 22 in the nascent biosynthetic
precursor (Fig. 2). This fact indicates that 1) the signal
sequence of porcine SPAI consists of 21 amino acids and 2) the plasma
species is an unprocessed form of the precursor proSPAI and contains
the complete sequence of the repetitive domain (Fig. 3) and
SPAI.
Figure 7:
Purification of plasma SPAI-like
immunoreactivity (SPAI-LI) and its identification as proSPAI by
N-terminal amino acid sequencing. A, a typical elution profile
of SPAI-LI from a reverse-phase C
HPLC column. SPAI-LI was
first extracted with a silica cartridge from porcine plasma (purity,
< 0.1%; yield, 80%) and partially purified with an antibody column
(purity, 25%; yield, 55%). The affinity-purified SPAI-LI was then
loaded onto the HPLC column and eluted by a CH
CN gradient.
One major and one minor peaks of SPAI-LI were emerged and termed
SPAI-LI
and SPAI-LI
, respectively, as indicated
by shaded boxes. About 2 nmol or 37 µg of SPAI-LI
was obtained from 1 liter of plasma with a yield of 43%
(estimated by ELISA); normal plasma levels of porcine SPAI were
1-5 nM. Although plasma samples did not contain low
molecular weight SPAI species (i.e. SPAI-1, SPAI-2, and
SPAI-3), their elution positions are also indicated by arrows. B, the N-terminal amino acid sequence of SPAI-LI
determined by direct sequencing. Initial sequencing attempts
failed probably because of the modification of the N terminus. We
therefore determined the sequence after removal of the N-terminal
pyroglutamate with pyroglutamyl peptidase. The sequence analysis and
alignment with the deduced amino acid sequence (Fig. 2)
indicated that the SPAI-LI
purified from porcine plasma
begins at Gln
as indicated by numbers in parentheses. The amount of SPAI-LI
was not
sufficient for sequencing.
Transglutaminase-mediated Cross-linking of
ProSPAI
To demonstrate that the N-terminal repetitive sequence
of proSPAI serves as a substrate for transglutaminase, we expressed and
purified proSPAI using the MBP fusion protein system (New England
Biolabs), and examined whether it is cross-linked by transglutaminase.
Highly purified proSPAI migrated as a closely spaced doublet on
SDS-PAGE (Fig. 8, lane 2); the upper band could be
converted to the lower band by increasing the incubation time with
Factor Xa, which was used to cleave proSPAI from the MBP-proSPAI fusion
protein. Furthermore, both bands reacted with anti-SPAI antibodies,
indicating that the doublet represents two forms of the same protein
probably generated by limited proteolysis during purification. When
treated with transglutaminase, proSPAI was readily cross-linked and
yielded higher molecular weight species (Fig. 8, lane
3); under the same experimental conditions, MBP and mature SPAI,
which lacks the repetitive sequence, were not cross-linked.
Figure 8:
Covalent cross-linking of proSPAI by
transglutaminase. Purified proSPAI was dialyzed against 5 mM Tris-HCl, pH 7.5, to remove phosphate, and aliquots (10 µg in
25 µl) were incubated in 10 mM Tris-HCl, pH 7.5,
containing 4 mM CaCl
and 8 mM dithiothreitol with (lane 3) or without (lane 2)
20 microunits of guinea pig liver transglutaminase (TGase,
Sigma) at 37 °C for 5 min, and subjected to SDS-PAGE. Sizes of
molecular markers (lane 1) are shown on the left in
kDa.
DISCUSSION
We have isolated and characterized a full-length cDNA and
genomic DNA clones for porcine SPAI-2. The amino acid sequence deduced
from the nucleotide sequence coincided with that previously determined
by direct sequencing of purified SPAI(1) , validating our
previous work. The presence of a hydrophobic presequence indicates that
SPAI-2 is a secreted protein. The presence of SPAI in the plasma
matches this secretory nature. Northern blot analysis indicated that
SPAI-2 mRNA is essentially confined to small intestine (Fig. 6).
Taken together, these results indicate that although the major site of
action of SPAI-2 is probably the intestine, it can be delivered to many
other tissues through the circulation.
The sequence comparison
between the ``mature'' SPAI and its precursor revealed that
the precursor is cleaved at the Asp
-Pro
bond ( Fig. 2and Fig. 4C). The Asp-Pro
bonds in proteins, however, have been demonstrated to be unstable under
strongly acidic conditions(15, 16) . The fact that the
mature SPAI was isolated from duodenum extracts exposed to such acidic
conditions(1) , therefore, strongly suggests that the 61-amino
acid SPAI might occur as an artifact during purification. In support of
this possibility, we could detect only proSPAI in plasma, suggesting
that the proSPAI is not a precursor of SPAI, but rather it is the
native form of SPAI.
A wide variety of proteins have been shown to
have the four-disulfide core structure and constitute the WAP protein
superfamily. They include neurophysins(17) , plant
agglutinin(3) , adhesion molecules(18) , whey
proteins(19, 20, 21, 22) ,
proteinase inhibitors(6, 23, 24) , scorpion
toxins (25) , bactericidal peptides(26) , pollen
proteins(27) , and SPAI(1) . So far the gene structures
of the following three members have been determined:
neurophysins(28, 29, 30) , mucous proteinase
inhibitor MPI(9) , and elafin(10) . The structure of
the SPAI-2 gene determined here is very similar to that of elafin (10) including the intron positions, indicating that the two
genes have arisen by a gene duplication. The genes for other members
might also have arisen from a common ancestor gene by exon and gene
duplications followed by gene conversion (28, 29) and
intron insertion(9) .
It is interesting that SPAI has a
repetitive sequence in its N-terminal region (Fig. 3) that is
very similar to that found previously in the human elafin
precursor(10) , whose sequence characteristics (i.e. rich in Gln and Lys) led us to propose that the repetitive
sequence serves as a substrate for transglutaminase, an enzyme that
catalyzes the formation of N
-(
-glutamyl)lysine isopeptide
cross-links between proteins(31, 32) . This was soon
confirmed to be the case by Molhuizen et al.(33) and
by ourselves(11) , establishing that elafin consists of two
domains: the transglutaminase substrate domain and the elastase
inhibitor domain. The transglutaminase substrate domain serves as an
anchor to localize elafin covalently to specific sites on extracellular
matrix proteins; therefore, we termed the transglutaminase substrate
domain of elafin the cementoin moiety(11) . The striking
similarity between the repetitive sequences of elafin and SPAI suggests
a similar role for the SPAI N-terminal extension; indeed, cross-linking
experiments indicated that the SPAI repetitive sequence is a good
substrate for transglutaminase (Fig. 8). The chances of the
interaction between the anchored SPAI and
Na
,K
-ATPase seem, however, very small
since the enzyme is a membrane protein and its movement is restricted.
The possibility, therefore, remains that SPAI, especially the anchored
SPAI, might have other as-yet-unidentified major targets of soluble
nature. The structural similarity between SPAI and elafin suggested
that SPAI is also a proteinase inhibitor; we therefore examined its
effects on the following proteinases by the standard assays using
chromogenic or fluorogenic synthetic substrates, but no inhibitory
activity was observed even at 5 µM: leucocyte elastase,
leucocyte chymotrypsin-like proteinase (cathepsin G), pancreatic
elastase, trypsin,
-chmotrypsin, and plasmin. This result
indicates that, at least, SPAI is not the porcine homolog of human
elafin. In support of this conclusion, we have recently identified a
SPAI analog in porcine trachea that is much similar to human elafin
than SPAI, and chemically synthesized it. The chemically synthesized
porcine SPAI analog (probably porcine elafin) inhibited pancreatic
elastase effectively (IC
= 50 nM) but
exhibited no Na
,K
-ATPase inhibitor
activity. (
)
In the present study, we determined the
structure, properties, and tissue distribution of porcine SPAI by cDNA
and gene cloning and Northern blot analysis. The interesting features
revealed are the presence of the transglutaminase substrate domain in
the SPAI sequence, the preferential localization of the SPAI message in
the intestine, and the presence of higher molecular weight form of
SPAI, which is considered to be the native form, in the circulation.
The information may form a valuable basis for elucidating, for example
by gene targeting, the physiological significance of SPAI.