From the
A new human leukocyte elastase inhibitor was extracted and
purified from a Korean native leech Hirudo nipponia. The
inhibitor, called guamerin, has a molecular weight of 6,110 and shows
inhibition constant (K
Human leukocyte elastase is a serine proteinase capable of
degrading important connective tissue proteins such as elastic fiber
(1) and collagen
(2) . It is stored in the cytoplasmic azurophil
granules and released into tissues when the cell encounters necrotic
tissues or microorganisms to be phagocytized
(3) . After
uncontrolled liberation at sites of inflammation, human leukocyte
elastase may cause nonspecific proteolysis and trigger destructive
processes associated with various chronic diseases including psoriasis
(4) and emphysema (5). The functional activity of human
leukocyte elastase is, in most cases, controlled by the presence of the
inhibitors
(6) . There has been growing interest in isolation and
characterization of properties of leukocyte elastase inhibitors because
the inhibitors may be used as therapeutic agents to prevent chronic
inflammatory diseases
(7) .
Elastase inhibitors have been
purified from human skin
(8, 9) , parotid
secretions
(10) , and bronchial mucus
(11) , as well as
from other sources such as soybean
(12) , turkey
ovomucoid
(13) , and leech
(14) . Eglin C is, in
particular, a potent elastase inhibitor isolated from Hirudo
medicinalis and characterized as a cysteine-free low molecular
weight protein
(14) . We hypothesized that Hirudo nipponia might also contain inhibitors that may have therapeutic
application to control processes associated with elastases.
In this
paper, we describe a new human leukocyte and porcine pancreatic
elastase-specific inhibitor, named guamerin, purified from a Korean
native leech H. nipponia. Its complete amino acid sequence was
determined, and the specificities against other proteases were
examined. We have found identical spacing of cysteine residues as
antistasin type serine-proteinase inhibitors, but the reactive site P1
residue is a methionine instead of arginine that determined specificity
of the inhibitor. To our knowledge, guamerin is the first cysteine-rich
elastase inhibitor isolated from leeches.
Assay for inhibitory activity against thrombin and factor Xa
was done essentially as described above.
Tos-Gly-L-Pro-L-Arg-pNA and
Bz-L-Val-Gly-L-Arg-pNA were used as substrates for
thrombin and factor Xa, respectively. The buffers used were 10
mM HEPES, pH 8.0, for thrombin and 0.2 M Tris-Cl, pH
8.0, containing 2.5 mM CaCl
The inhibitory activity of guamerin on the digestion of azocasein by
the other proteases was measured using an assay mixture that included
150 µl of 50 mM Tris-Cl, pH 8.0, 50 µl of enzymes
(from 0.05 to 0.2 mg/ml), 40 µl of guamerin (1.75 µM)
or buffer (serving as control). In the case of papain and pepsin, 0.2
M Tris-Cl, pH 8.0, containing 1 mM cysteine, 20
mM EDTA, and 10 mM sodium acetate, pH 5.4, were used
as reaction buffers, respectively. Upon the addition of 0.5 ml of 0.1%
(w/v) azocasein in the same buffer, the tubes were incubated at 37
°C for 50 min. The reaction was terminated by adding 0.5 ml of 15%
trichloroacetic acid, and the tubes were allowed to stand on ice for 15
min. The precipitate was removed by centrifugation, and the absorbance
at 440 nm was measured
(18) . The percentage of inhibition was
calculated using the above equation.
The amino acid and sequence analyses of
native guamerin were done by the Pico-Tag method (Waters) after
hydrolysis in constant boiling HCl (Sigma) at 110 °C for 24 h. In
one sample, cysteine residues were oxidized to cysteic acid with a
mixture of formic acid and hydrogen peroxide (19:1, v/v)
(20) .
To determine the tryptophan content, guamerin was directly digested
with 20 µl of 4 N methanesulfonic acid
(21) . Amino
acid sequences of peptides were analyzed in an Applied Biosystems 476A
protein sequencer. Prior to peptide digestion, the N terminus was
investigated by automated Edman degradation.
The complete amino acid sequence data are
given in I. Following reduction and
S-pyridylethylation of guamerin, direct sequencing gave 57
residues and a blocked N terminus was not found. The total sequence was
confirmed by overlapping the sequences of fragments digested with
trypsin or endoproteinase Lys-C. When S-pyridylethylated
guamerin was digested with endoproteinase Lys-C followed by separation
of the peptides with a Delta-Pak C
We have purified an elastase-specific inhibitor, guamerin,
from H. nipponia and determined its complete amino acid
sequence. Purification of guamerin was done by stepwise gel filtration,
anion exchange, and reversed phase HPLC. The inhibitory peptides from
DEAE-anion exchange chromatography were separated with an HPLC
gradient, producing four peptide peaks with anti-elastase activity
(Fig. 1C). During the purification and sequencing of
guamerin, three more active peaks were observed (in
Fig. 1C, designated as peaks 1, 2, and
4) with a dual gradient of acetonitrile elution. Their
molecular weights were 5734, 7800, and 6212, respectively. Analysis of
the second most active peak (peak4) showed over 90%
homologous amino acid sequence as well as identical cysteine spacing
and the same P1 residue as guamerin. This suggests that it must be an
isotype of guamerin. The rest of the active fractions will be further
characterized to find sequences and variabilities.
When sequence
similarity was studied, guamerin showed no homology to any known
elastase inhibitors including eglin C and elafin but had 51% sequence
identity to hirustasin, antistasin-type serine proteinase inhibitor
from H. medicinalis and 39% sequence similarity to antistasin
(domain-2), a factor Xa inhibitor, from the saliva of a Mexican leech
Haementeria officinalis (Fig. 5). Neither hirustasin nor
antistasin is, however, known for inhibitory activity against
elastases
(25, 26) . In addition to conservation of
cysteine residues with identical spacing, it is a striking feature that
octapeptide (
In conclusion, guamerin is similar in both size and cysteine content
to hirustasin, an antistasin-type serine protease inhibitor isolated
from leeches, but it has a different target proteinase with a specific
reactive site. It appears that guamerin is an elastase-specific
inhibitor and differs from previously characterized elastase inhibitors
in primary structure and K
Approximate yields of phenylthiohydantoin (PTH)-derivatives
were calculated from peak areas on HPLC.
We thank Dr. Jin Sung Kim for MALDI-MS analysis, Soo
Hyun Kim and Jong Soon Choi for computer facilities, and Dr. In Jung
Yoon and Jae Hie Shim for helpful discussions.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
) of 8.1
10
M. It is stable at a wide range of pH
from 1 to 11 and heat-stable up to 90 °C. The complete amino acid
sequence of guamerin reveals a cysteine-rich polypeptide of 57 amino
acid residues that shows no similarity to any known elastase inhibitors
but has 51% sequence homology with hirustasin. Guamerin has identical
spacing of 10 cysteine residues as antistasin-type serine proteinase
inhibitors, but the P1 reactive site residue is Met
instead of Arg. The neighboring sequence of the reactive site
consists primarily of hydrophobic amino acid residues. Based on
examinations of the target proteinases and the reactive site
specificity, guamerin is a new low molecular weight protein that
inhibits elastases.
Materials
Adult leeches were collected from a
lake at Chonju, cultured in the laboratory for 6 months
(15) ,
and starved for 2 weeks prior to being utilized. Human leukocyte
elastase, porcine pancreatic elastase, bovine pancreatic trypsin,
-chymotrypsin, subtilisin, pepsin, papain, bovine plasma factor
Xa, bovine plasma thrombin, azocasein, 4-vinylpyridine, and all
chromogenic substrates were purchased from Sigma. Endoproteinase Lys-C
and trypsin (sequencing grade) were provided by Boehringer Mannheim.
Sephadex G-75 and DEAE-Sepharose were purchased from Phamarcia Biotech
Inc. Sep-Pak C
cartridge and Delta-Pak C
reversed phase HPLC
(
)
columns were obtained
from Waters division of Millipore.
Purification of Guamerin
Starved leeches (40 g)
were minced and ground on ice, and 200 ml of 80% (v/v) of cold aqueous
acetone was added with stirring at 4 °C for 10 min. Then, sodium
chloride and trichloroacetic acid were added to final concentrations of
0.3 and 0.4 M, respectively, with constant stirring for 60
min. The insoluble residue was removed by centrifugation at 6,000 rpm
for 10 min. The resulting supernatant was mixed with 4 volumes of 100%
ice-cold acetone to precipitate elastase inhibitory peptides and
centrifuged at 12,000 rpm for 20 min. The pellet was suspended in 50
mM Tris-Cl, and about 5% of the pellet was solubilized in the
buffer. No elastase inhibitory activity was found in the insoluble
part, which was removed by centrifugation at 10,000 rpm for 20 min. The
solution was applied to a Sephadex G-75 column (110 2 cm)
equilibrated with 50 mM Tris-Cl buffer, pH 8.0 containing 200
mM NaCl and eluted with the same buffer at a flow rate of 0.5
ml/min. The inhibitor fractions were pooled, desalted, and then applied
to a DEAE-Sepharose column (7
1.6 cm) equilibrated with the
same buffer and eluted with a 60-ml linear gradient of NaCl from 0 to
400 mM. The inhibitor fractions were pooled, concentrated, and
desalted with a Sep-Pak C
cartridge. The desalted fraction
was applied to a Delta-Pak C
reversed phase HPLC column
(3.9
300 mm) equilibrated with buffer A and eluted with a
linear gradient composed of buffer B. Buffer A is 0.1% trifluoroacetic
acid, and buffer B is 80% acetonitrile containing 0.1% trifluoroacetic
acid. Four inhibitory peptides were separated at a range of
25-60% of buffer B. The inhibitor fractions were separately
collected, dried, and stored at -20 °C until use.
Assay of Inhibitory Activity
The anti-elastase
activity of the purified peptide was estimated from the residual
activity of elastase in the mixture of peptide and the protease. The
standard assay mixture contained 40 µl of 0.2 M Tris-Cl,
pH 8.0, containing 20 µl of 0.37 mg/ml human leukocyte elastase and
either 40 µl of guamerin (1.75 µM) or 40 µl of
buffer. The reaction was initiated with the addition of 400 µl of
succinyl-Ala-Ala-Ala-p-nitroanilide (from 0.2 to 1.6
mM) in the buffer. After incubation at 37 °C for 1 min the
reaction was stopped by placing on ice. The appearance of
p-nitroaniline was spectrophotometrically monitored at 410
nm
(16) . The percentage of inhibition (%I) was calculated as %I
= [(1 -
A /A
)]
100, where A
and
A
are the absorbance with and without
guamerin, respectively. The inhibition constant
(K
) of guamerin for the hydrolysis of
Suc-Ala-Ala-Ala-pNA was determined according to the method of Dixon
(17).
to activate factor Xa.
Reduction and S-Pyridylethylation
Guamerin (80
µg) dissolved in 200 µl of 0.1 M Tris-Cl pH 8.0
containing 6 M guanidine hydrochloride was mixed with 10
µl of 2-mercaptoethanol. The mixture was flushed with nitrogen gas
and then incubated at 50 °C for 7 h. The free sulfhydryl groups
were exposed to 15 µl of 4-vinylpyridine, and the solution was
stirred at room temperature in the dark for 2 h
(19) . The
reduced and S-pyridylated guamerin was then repurified on a
Dalta-Pak C column (3.9
300 mm) by 0-50%
gradient of acetonitrile in 0.1% trifluoroacetic acid at a flow rate of
1 ml/min and used for subsequent analyses.
Enzymatic Cleavage of S-Pyridylethylated
Guamerin
S-pyridylethylated guamerin (40 µg, 13
nmol) was digested with trypsin (2 µg) in 100 µl of 0.1
M Tris-Cl buffer, pH 8.0, at 37 °C for 20 h. In a separate
reaction, this peptide (40 µg) was also digested with
endoproteinase Lys-C (1.5 µg) in the same buffer at 37 °C for
24 h. The reaction was stopped by freezing on dry ice.
Isolation of the Peptides after Digestion
Peptide
fragments resulting from enzymatic digestion were purified by a Waters
HPLC system (510 HPLC pump, 717 plus automatic sampler, 996 photodiode
array detector, and Millennium 2010 chromatography manager) equipped
with a Delta-Pak C reversed phase column (3.9
300
mm) equilibrated with buffer A. Peptides were eluted from the column
with a linear gradient of buffer B (0-50%, 0.42%/min) at a flow
rate of 0.5 ml/min at 25 °C. Absorbance at 214 nm was measured, and
peptides were manually collected for sequence analysis. Peptides were
numbered according to the order of elution from the HPLC column.
Peptides derived from trypsin and endoproteinase Lys-C were designated
as T and L, respectively.
Molecular Weight Determination by Matrix-assisted Laser
Desorption Ionization Mass Spectrometry (MALDI-MS)
One µl of
guamerin (6.1 mg/ml) dissolved in aqueous 0.1% trifluoroacetic acid was
mixed with 2 µl of sinapinic acid (70 g/liter). The mixture was
loaded on a flat inert metal (e.g. silver, platinum) probe.
The solvent was removed in a stream of warm air, and the sample was
then transferred to the vacuum chamber. Thus the typical absolute
sample amount used for analysis was about 10 pmol. Then the molecular
weight of guamerin was detected by MALDI-MS
(22) . Bovine
pancreas insulin (M 5,734) and cytochrome c (M
12,328) were used as reference peptides to
calibrate the molecular weight of guamerin exactly.
Purification of Guamerin
Crude extract from
leeches was mixed with 50 mM Tris-Cl buffer, pH 8.0, and
centrifuged to remove insoluble materials. Approximately 95% of
nonactive proteins was removed as insoluble pellet. The supernatant
enriched in anti-elastase activity was applied to a Sephadex G-75
column equilibrated with 50 mM Tris-Cl buffer, pH 8.0,
containing 200 mM NaCl. Elastase inhibitory
activity-containing fractions from the Sephadex G-75 column were
pooled, desalted (Fig. 1A), and fractionated further by
passage over a DEAE-Sepharose column preequilibrated with 50
mM Tris-Cl buffer, pH 8.0. The column was developed with a
linear NaCl gradient from 0 to 400 mM, and inhibitory activity
was eluted as a peak at around 150 mM NaCl
(Fig. 1B). Following desalting and acidifying, the
active fractions from DEAE-Sepharose were subjected to reversed phase
C HPLC (Fig. 1C). The most activity was
found as a major peak in Fig. 1C, designated as peak3. Its purity was confirmed by three different methods.
1) Tricine-SDS-polyacrylamide gel electrophoresis
(23) showed a
single band (data not shown); 2) MALDI mass spectrum provided a single
sharp peak with a molecular weight of 6,110 (Fig. 2); and 3) gas
phase protein sequencing has detected a single amino acid on the N
terminus. The results of each purification of guamerin are shown in
.
Figure 1:
Purification steps of guamerin by
sequential chromatography. A, Sephadex G-75 column profile.
The inhibitory activities against human leukocyte elastase were found
at fractions 30-40; B, DEAE-Sepharose column profile.
The active fractions 16-20 were desalted and concentrated. The
dottedline indicates a linear salt gradient from 0
to 400 mM; C, Dalta-Pak C HPLC column
profile. Peptides were eluted with a linear gradient of acetonitrile.
The active peaks were numbered based on the order of elution. The most
active peak (peak3) was isolated in bulk and assayed
subsequently. The remaining active peaks are mentioned under
``Discussion.''
Figure 2:
MALDI mass spectra of guamerin and
reference substances. The arrow shows the molecular weight
(6,110) of guamerin. This value was confirmed to be correct by the
result calculated from amino acid sequence analysis. The peak at
mass/charge 12328.6 and 5734.5 indicates the molecular weight of
cytochrome c and insulin,
respectively.
Physicochemical Properties
The stability of
guamerin at elevated temperatures was investigated. When guamerin was
incubated in 0.1 M Tris-Cl buffer pH 8.0 from room temperature
to 90 °C at 10 °C intervals for 15 min each no loss of the
inhibitory activity was observed. Guamerin was also stable in a wide
range of pH from 1 to 11 at 25 °C. Guamerin competitively inhibited
the catalytic activity of elastase in a substrate-like manner in the
presence of the low molecular weight synthetic peptide substrate
(Suc-Ala-Ala-Ala-pNA) as well as a high molecular weight substrate
(azocasein). This result was obtained by using human leukocyte elastase
with a specific activity of 100 units/mg protein and 0.17
µM of guamerin according to the method of Dixon (17). The
inhibition constant of guamerin was 8.1 10
M, which is considerably lower than eglin C
(10
10
M) or
elafin (6
10
M)
(9, 14) . In addition, when the enzyme
and guamerin were preincubated for varying time intervals prior to
adding the substrate to reaction vials, no differences in inhibitory
activities were found, suggesting that guamerin is a fast binding and
slow dissociating molecule.
Amino Acid Composition and Sequence Analysis
The
amino acid composition of guamerin was determined by Pico Tag amino
acid analysis, and the result is shown in . The number of
residues/molecule analyzed by hydrolysis was based on a molecular
weight of 6,110, which was identical to the result obtained from the
complete sequence of the peptide. It is very interesting that guamerin
consists of 10 cysteines and no tryptophan. The significance of a large
number of cysteine residues is unclear except that they play a pivotal
role in structural rigidity. The importance of disulfide bridges was
confirmed by a subsequent experiment showing that reduction and
S-pyridylethylation caused loss of inhibitory activity of
guamerin (data not shown).
reversed phase HPLC
column, three peptide fragments (L1, L2, and L3) were obtained. Tryptic
digestion produced four peptide peaks (T1, T2, T3, and T4) and a ghost
peak containing no peptide under the same condition (data not shown).
The alignment of all the tryptic and lysyl endoproteinase peptides is
shown in Fig. 3.
Figure 3:
Amino acid sequencing strategy of
S-alkylated guamerin. The direct sequence was determined by
automated Edman degradation without fragmentation. The bars represent the overlapping fragments derived from enzymatic
cleavage of trypsin (T) and endoproteinase Lys-C (L).
Peptides were numbered according to the order of elution from HPLC
column after enzymatic digestion (data not
shown).
The specificity of guamerin was studied with
various kinds of proteases, which are known to have different active
site residues. The molar ratio of guamerin to each enzyme was adjusted
to 5. Guamerin inhibited human leukocyte and porcine pancreatic
elastase by 86.4 and 83.2% on average, respectively (Fig. 4).
However, guamerin had a very small effect on trypsin (serine protease),
chymotrypsin (serine protease), and subtilisin (serine protease) and no
effect on papain (thiol protease), pepsin (aspartyl protease), thrombin
(serine protease), and factor Xa (serine protease). These results show
that guamerin may be an elastase-specific inhibitor. It has been known
that the reactive center(
)
P1-P1` peptide bond of
the inhibitor may be cleaved by its target protease and the peptide
bond determines the specificity
(24) . We attempted to examine
which site(s) within guamerin are cleaved by human leukocyte elastase.
When guamerin-human leukocyte elastase complex was subjected to
automated protein sequencing after incubation for 48 h, we observed
that the Met
-Ile
peptide bond within guamerin
was hydrolyzed by elastase.
Figure 4:
Inhibition of various proteinases by
guamerin. Guamerin was effective for human leukocyte and porcine
pancreatic elastase but had no significant effect on subtilisin,
chymotrypsin, trypsin, papain, pepsin, factor Xa, and thrombin. The
bar represents standard deviation of four separate
experiments. The experimental conditions are described under
``Experimental Procedures.'' HL-Elastase, human
leukocyte elastase; PP-Elastase, porcine pancreatic
elastase.
TCSPAQVC
) and nonapeptide
(
DENGCEYPC
) are completely conserved in
guamerin and hirustasin (Fig. 5). These homologies suggest that
guamerin, hirustasin, and antistasin may have a common ancestor and
that gene duplication and mutation events may be sources of diversity
in these inhibitors
(26, 27) . Guamerin is a good example
of the case that even though it has a highly homologous sequence to
hirustasin and antistasin, its target protease is different from them.
The specificity of the inhibitor is generally determined by the P1
residue of the reactive site
(24, 28) . In hirustasin and
antistasin, Arg is identified as the reactive site
residue
(25, 26, 27, 29, 30) .
The sequence alignment indicates that the P1 residue Met
of guamerin corresponds to the Arg
residue at the P1
position for hirustasin and to Arg
, Arg
for
two domains of antistasin (Fig. 5). Thus, we can suggest that the
mutational replacement of Met for Arg at the reactive site P1 of
guamerin resulted in the change of its activity. The hydrophobic region
surrounding the reactive site is known to be preferred for human
leukocyte elastase
(31) . From Kyte-Doolittle hydropathy profiles
(32) we have evidence that the secondary structure of guamerin
had a different environment around the reactive domain from hirustasin
and antistasin domain-2. The reactive domain of guamerin lies in a
hydrophobic region, whereas the reactive domain of the others are
hydrophilic. In addition to the distinct reactive site, we suggest that
hydrophobic groups around the reactive site play a role in the
specificity of guamerin.
Figure 5:
Amino acid sequence homology of guamerin
with hirustasin and antistasin. Dark boxes show matches of
cysteine residues, and the shadedboxes indicate
homologous sequence. The arrow marks the scissile peptide bond
(the reactive center) of the three inhibitors. The sequence of elafin,
an elastase-specific inhibitor of human skin with 57 amino acid
residues, has virtually no homology.
There are a few specific elastase
inhibitors having a similar molecular mass. For example, elafin, a
human leukocyte elastase-specific inhibitor from human skin, has 57
amino acid residues but has virtually no sequence homology
(Fig. 5). The preliminary data from ingestates suggested that
guamerin was not present in the saliva of leeches but in the body
tissue, perhaps implying a primary role in regulation of tissue
proteinases. Further studies including cloning of cDNA are necessary to
ascertain structural and functional properties of this interesting
protein with the long range goal of application as a therapeutic agent.
value.
Table:
Purification of guamerin
Table:
Amino acid composition of guamerin
Table:
Automated sequence analysis of S-alkylated
guamerin
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.