From the Department of Biochemistry and Molecular Biology,
University of Georgia, Athens, Georgia 30602
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INTRODUCTION |
Asthma is an allergic inflammation of the lungs which can occur
after allergen sensitization. Such inflammatory responses are normally
meant to defend against invading organisms or particulates or to effect
tissue repair and are thus beneficial; however, in asthma, the response
becomes exaggerated (perhaps because of a hereditary predisposition
(1)), leading to adverse effects on the airways (2). Macrophages
phagocytize the allergens introduced to the lungs by exposure to
various environmental irritants such as dust, pollutants, and pollen,
and process them to smaller fragments. As antigen-presenting cells,
they then activate T-cells (3, 4) to stimulate B-cells to produce IgE.
This immunoglobulin, when bound to a specific allergen, in turn,
stimulates and activates several alveolar cell types to produce the
many mediators of inflammation: histamine, prostaglandins,
leukotrienes, cytokines, neutral proteases, active oxygen species, and
chemoattractants (5). The interaction of these mediators leads to the
pathology of asthma, including bronchoconstriction, hypertrophy of
airway smooth muscle, vasodilation, submucosal edema, and mucus
hypersecretion (6). Also, the mucociliary apparatus becomes
dysfunctional, reducing the clearance of inhaled particulates.
Epithelial cells lining the airways are shed during this inflammatory
response, removing a protective barrier (2) and are also a source of
neutral endopeptidase (which normally degrades various
bronchoconstrictor peptides (7)) while exposing nerve endings (8) that
secrete neuropeptides such as vasoactive intestinal peptide
(VIP)1 and substance P, and
vasoactive peptides (e.g. angiotensin II). VIP, a
neurotransmitter of the nonadrenergic inhibitory system (9), has an
anti-inflammatory effect inhibiting lymphocyte proliferation and
interleukin-2 release and is also a potent bronchodilator (10).
Substance P, a neurotransmitter of the nonadrenergic excitatory system
(11), in contrast, has a proinflammatory effect, increasing vascular
permeability and bronchoconstriction, causing macrophages to release
proinflammatory substances, and enhancing phagocytosis by neutrophils
and macrophages (12). Angiotensin II is a strong vasoconstricting agent
(13).
Pollen is one of the major initiators of allergic asthma. This gamete
contains proteins (allergens) that are solubilized in the airway mucus
and proceed to induce an immunological response. However, other
proteins are also released, of which several have proven to be
oligopeptidases (14-16). Because these latter enzymes appear to be
members of a family of pollen oligopeptidases with varying
specificities for peptide hydrolysis, we propose to name them, at least
temporarily, as: peptidases Imes and Irag
(trypsin-like specificity from both mesquite and ragweed pollens),
peptidase IIrag (chymotrypsin-like specificity from ragweed
pollen), and, as described in this report, peptidase IImes
(hydrophobic amino acid specificity from mesquite pollen), an enzyme
that rapidly degrades VIP, angiotensin II, and its precursor,
angiotensin I. We suggest that through exo- and oligopeptidase
activity, pollen may have the capability for participation in the
inflammatory processes in allergic asthma by mechanisms other than
those involving its immunological component.
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EXPERIMENTAL PROCEDURES |
Materials
H-Val-pNA, H-Leu-pNA,
N-Suc-Ala-Ala-Pro-Phe-pNA,
N-Suc-Ala-Ala-Pro-Leu-pNA,
N-Suc-Ala-Ala-Val-Ala-pNA,
N-Suc-Ala-Ala-Ala-pNA, N-Suc-Phe-pNA,
benzoyl-DL-Arg-pNA, TPCK, TLCK, iodoacetamide, bestatin
([(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl]-L-leucine), angiotensins I and II, VIP, atrial natriuretic peptide, bradykinin, substance P, neurotensin, Phe-Gly-Leu-Met (substance P fragment) (peptide 1), Phe-Ser-Trp-Gly-Ala-Glu-Gly-Gln-Arg (active fragment of
myelin basic protein) (peptide 2), Ala-Ser-Thr-Thr-Thr-Asn Tyr-Thr
(peptide T = HIV inhibitor) (peptide 3), and Leu-Pro-Pro-Ser-Arg (lymphocyte-activating pentapeptide from the Fc region of
human IgG1) (peptide 4) were obtained from Sigma. H-Ala-pNA,
H-Ala-Ala-pNA, H-Ala-Ala-Ala-pNA,
Ac-Ala-pNA, Ac-Ala-Ala-pNA, H-Phe-pNA,
H-Ile-pNA, H-Ala-Phe-pNA,
H-Glu-Ala-pNA,
N-Suc-Ala-Phe-Pro-Phe-pNA,
Suc-Ala-Ala-Pro-Ala-pNA, benzyloxycarbonyl-Ala-Ala-Leu-pNA, and
benzoyl-Tyr-pNA were from Bachem. Diisopropyl
fluorophosphate and 3,4-dichloroisocoumarin were obtained from
Calbiochem, and AEBSF and EDTA were from Boehringer Mannheim. The
mesquite pollen was a kind gift from Dr. Justin O. Schmidt (Carl Hayden
Bee Research Center, Tuscon, AZ). All chloromethyl ketone (except TPCK
and TLCK) and organophosphonate inhibitors were kindly provided by Dr.
James Powers (Georgia Institute of Technology, Atlanta).
Methods
Enzyme Extraction and Purification--
Mesquite pollen (100 g)
was extracted by stirring in 400 ml of 0.02 M Bis-Tris, pH
6.5, 5 mM CaCl2 (buffer A) overnight at 4 °C. Purification of the enzyme was performed using exactly the procedures described previously (14) with ammonium sulfate
fractionation, acid precipitation of contaminants, and Cibacron
blue-Sepharose, DEAE-Sephacel, and phenyl-Sepharose chromatography. The
active eluate from the phenyl-Sepharose column was dialyzed overnight at 4 °C against buffer A with two changes and concentrated to 20 ml
using an Amicon P-30 membrane. The final step of purification involved
the application of the dialyzed and concentrated enzyme solution to a
Mono Q FPLC column (Amersham Pharmacia Biotech) equilibrated with
buffer A. The column was washed with buffer A for 5 min, followed by a
0-0.05 M NaCl gradient for 5 min, then a 0.05-0.15
M NaCl gradient for 50 min during which the enzyme activity
was eluted. The native conformation of the enzyme was obtained by
polyacrylamide gel electrophoresis using a Tris-HCl/Tricine buffer
system (17) omitting SDS.
Molecular Weight Determination--
The molecular weight of the
purified enzyme (peptidase IImes) was determined by both
SDS-polyacrylamide gel electrophoresis using a Tris-HCl/Tricine buffer
system (17) with or without reducing conditions and by gel filtration
on a Sephadex G-150 column (2.2 × 90 cm).
Enzyme Assays--
For routine assays during purification, pH
optimum determination, temperature effects, and the effects of
inhibitors, the activity of peptidase IImes was only
measured spectrophotometrically at 405 nm with H-Ala-pNA (1 mM, final concentration) in either 0.2 or 1.0 ml of 0.1 M Tris-HCl, pH 8.0, 0.15% dimethyl sulfoxide at 25 °C.
In inhibitor studies, the enzyme was incubated with inhibitors for 15 min at 25 °C before the substrate (H-Ala-pNA) was added.
Amidolytic activity of several substrates (1 mM, final concentration) was determined in 0.2 ml of the same buffer and temperature as above. Protein concentration was determined by the
bicinchoninic acid-Cu(II) sulfate procedure with bovine serum albumin
as the standard (18).
Sequence Analysis--
Peptidase IImes (1.06 nmol)
was denatured by boiling in 1% SDS followed by incubation with 0.017 nmol of high molecular weight Arg-gingipain from Porphyromonas
gingivalis (19) in 0.2 ml of 0.02 M Tris-HCl, pH 7.6, and 1 mM fresh cysteine overnight at 37 °C. After
SDS-polyacrylamide gel electrophoresis of the digest and electroelution
to a polyvinylidene difluoride membrane, sequence analysis was
performed with an Applied Biosystems Procise Protein sequencer using
the program designed by the manufacturer.
Enzyme Specificity and Kinetics--
For specificity studies,
the purified enzyme (35.3-106.0 nM) was incubated with
several bioactive peptides (20.0-64.0 µM) at
enzyme:substrate molar ratios of 1:400-1:600 in 0.1 M
Tris-HCl, pH 8.0, at 37 °C. For studies with peptides with
NH2-terminal residues of phenylalanine, alanine, and
leucine, the purified enzyme (58.7-78.0 nM) was incubated
with each of the substrates (64.3-143.2 µM) at
enzyme:substrate molar ratios of 1:1,000-1:8,000 in the same buffer
and temperature as above. Aliquots of 35 µl were removed at various
times and added to 2 µl of 20% trifluoroacetic acid to stop the
reaction. Each reaction mixture was subjected to high performance
liquid chromatography (HPLC) using an Ultrasphere ODS reverse phase
column (4.6 × 25.0 cm, 5 µm) (Beckman Instruments) and a linear
gradient from 0.1% trifluoroacetic acid to 0.08% trifluoroacetic acid
containing 80% acetonitrile over a 30-min period (1 ml/min). Peptides
were detected at 220 nm. The same reaction mixtures were analyzed for
amino acid composition by mass spectrometry. Some of the samples were
examined by matrix-assisted laser desorption ionization. The matrix (2 µl of a saturated solution of
-cyano-4-hydroxycinnamic acid in a
50:50 mixture of water:acetonitrile with 0.1% trifluoroacetic acid)
was placed on the target with approximately 0.5 µl of sample. The
samples were then analyzed with a Bruker Reflex time-of-flight mass
spectrometer (Billerica, MA) using matrix-assisted laser desorption
ionization in linear mode, 100 shots averaged with mass range scanned
from 0 to 1,500 m/z for bradykinin to 0-3,600
m/z for VIP. Some samples were analyzed by liquid
chromatography-mass spectrometry using a PE-Sciex API I (atmospheric
pressure ionization) plus mass spectrometer coupled with an Applied
Biosystems 140 B solvent delivery system and an ABI 759A absorbance
detector. The sample (20 µl) was injected onto an Asahipak ODP C18
column (1 × 250 mm, 5 µM, 200 A) (Keystone Scientific Inc., Bellefonte, PA). The gradient used was from 0 to 100%
B over 60 min at a flow rate of 40 µl/min. Solvent A was 0.1%
trifluoroacetic acid in water, and solvent B was 90% acetonitrile and
10% water with 0.1% trifluoroacetic acid. The mass spectrometer was
scanned from 50 to 500 U using a 0.2-U step and a 1.5-ms dwell time.
The UV was monitored at 214 nm, and the signal was amplified 50 times
by an Omni Amp II A (Omega Engineering Inc., Stamford, CT). The results
of the mass spectrometry were in the form of a chromatographic trace
with each peak having a number representing the mass of a specific
fragment. Each number was entered into the computer program, Peptidemap
2.2, together with the sequence of the peptide being studied. The
output obtained indicated the sequence of the peptide fragment matching
the mass of the peak and was an indication of cleavage products from
peptidase IImes proteolysis.
Vmax and Km values for amino
acid p-NAs were determined using substrate concentrations
ranging from 18.75 to 250 µM with the final
concentrations of enzyme from 2.0 to 19.1 nM in 0.1 M Tris-HCl, pH 8.0, 0.125% dimethyl sulfoxide at 25 °C. Values for the bioactive peptides were measured with substrate concentrations ranging from 10 to 82 µM, with the final
concentration of enzyme from 2.2 to 25.4 nM in 0.1 M Tris-HCl, pH 8.0, 5 mM CaCl2 at
25 °C with peptidase Imes and 37 °C with peptidase
IImes. Aliquots of 35 µl were removed at various times
and added to 2 µl of 20% trifluoroacetic acid to stop the reaction.
Each sample was subjected to HPLC as described above. The increase in
the peak area of the product with time was used to determine the rate of peptide cleavage. Vmax and
Km values were determined by using Hyperbolic
Regression Analysis.2
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RESULTS |
Enzyme Purification--
Peptidase IImes was readily
liberated from the pollen grains by gentle stirring with buffer at
4 °C, with 50% of the activity being released by 2.5 h, and
maximum activity at 6 h (data not shown). However, because the
enzyme was very stable, extraction was usually performed overnight as a
matter of convenience.
As shown in Table I, several steps were
required to purify peptidase IImes, with the scheme
utilized being essentially equivalent to that performed for the
isolation of peptidase Imes (14). Although a single enzyme
activity directed toward hydrolysis of H-Ala-pNA was
obtained during all procedures up to the Mono Q FPLC step, three
activities separated during this final chromatographic procedure.
However, all forms exhibited identical specific activities against
either H-Ala-pNA or H-Leu-pNA, all were 92 kDa,
and all were inhibited by TPCK, 3,4-dichloroisocoumarin, AEBSF, or the aminopeptidase inhibitor bestatin. A native polyacrylamide gel revealed
a single, diffuse, unresolvable band (data not shown). Because of these
identical properties, we assumed that the various forms were isozymes
of each other, pooled them together, and utilized the combined enzyme
in the studies described below.
As in the case of peptidase I mes, peptidase II
mes was also stable for at least several months at
20 °C, although frequent freezing and thawing caused some loss.
However, in comparison, Ca2+ was not required either for
stability or activity.
Physical Properties--
Treatment of the purified enzyme with SDS
followed by gel electrophoresis revealed a major band with a molecular
mass of 92 kDa and some very faint minor bands (Fig.
1). The molecular mass of the major band
agreed very well with that determined by Sephadex G-150 gel filtration
of active enzyme (96 kDa). Unfortunately, no amino-terminal sequence
could be found, indicating that this enzyme has a blocked amino
terminus. Utilizing the amidolytic activity assays with
H-Ala-pNA, it was found that the enzyme had a broad pH
optimum from pH 7.5 to 9.5 and was stable for at least 48 h at pH
8.0 and 25° or 37 °C.

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Fig. 1.
SDS-polyacrylamide gel electrophoresis of
mesquite pollen peptidase IImes at various stages of
purification. Lanes 1 and 9, molecular mass
markers (rabbit muscle phosphorylase b, 94 kDa; bovine serum
albumin, 67 kDa; ovalbumin, 43 kDa; bovine erythrocyte carbonic
anhydrase, 30 kDa; soybean trypsin inhibitor, 20 kDa; -lactalbumin,
14 kDa). The following lanes contained boiled and reduced samples:
lane 2, pollen extract; lane 3, ammonium sulfate
precipitate; lane 4, acid supernatant; lane 5,
Cibacron blue-Sepharose wash; lane 6, DEAE-Sephacel eluate;
lane 7, phenyl-Sepharose eluate; lane 8, Mono Q
FPLC eluate. The gel was stained with Coomassie Blue R-250.
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Amidase and Peptidase Specificities--
Peptidase
IImes activity was tested with several amino acid and
peptide p-NAs (Table II).
H-Ala-pNA was the preferred substrate by far (and thus was
used in general assays), with the next best being
H-Ala-Ala-pNA. Longer peptides were even less effective as
substrates. An NH2-terminal blocking group nearly or
completely abolished activity, with Suc-Ala-Ala-Ala-pNA,
Suc-Phe-pNA, Ac-Ala-pNA, and
Ac-Ala-Ala-pNA not acting as substrates at all; however,
there was substantial activity against the corresponding
non-succinylated or non-acetylated p-NAs.
Ac-Ala-pNA and Ac-Ala-Ala-pNA, in fact, acted as
inhibitors at 10 and 20 times the concentration of the substrate,
H-Ala-pNA. These results indicate that the amidolytic activity of peptidase IImes requires a free amino group at
the NH2 terminus of a substrate, whereas a blocked
NH2 terminus can create a competitive inhibitor. It is
possible that the enzyme may be sequentially removing the
NH2-terminal amino acid or cleaving internally in the
peptide pNA substrates since, as shown below utilizing
non-pNA peptide substrates, both aminopeptidase and oligopeptidase activity could be detected.
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Table II
Amidolytic activity of mesquite pollen peptidase IImes
The assay was performed at 25 °C in 0.1 M Tris-HCl, pH 8.0, 0.15%
Me2SO with 1 mM, final concentration, of the substrates above.
The enzyme to substrate ratio was 1:100,000 for H-Ala-pNA and 1:25,000
for the others.
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The demonstration of exo- and oligopeptidase activity of peptidase
IImes against both bioactive and randomly selected peptides is given in Table III. In peptides chosen
because they contained unblocked phenylalanine, leucine, or alanine
residues at the NH2 terminus, hydrolysis at the amino
terminus occurred at low E:S molar ratios: 1:8,000 for
enzyme:FGLM, 1:2,000 for LPPSR, and 1:1,000 for both FSWGAEGQR and
ASTTTNYT. Internal residues of alanine and leucine were untouched at
these short times of incubation and low E:S ratios. The
enzyme was particularly effective in cleaving after
NH2-terminal phenylalanine residues, especially in the
tetrapeptide, FGLM. Hydrolysis after either the
NH2-terminal leucine or alanine residues was much slower
(Fig. 2). Thus, peptidase
IImes exhibited aminopeptidase activity. It is puzzling,
however, why phenylalanine should be preferred rather than alanine, as
was seen with the pNA substrates.

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Fig. 2.
Decrease in percent of peak area of various
peptides with phenylalanine, leucine, or alanine in the
NH2-terminal position from HPLC after incubation with
mesquite pollen peptidase IImes. Purified enzyme was
incubated with FGLM, FSWGAEGQR, LPPSR, or ASTTTNYT in 0.1 M
Tris-HCl, pH 8.0, at 37 °C as described under "Methods." At
various times, aliquots were removed and acidified to stop the
reaction, and peaks were separated on HPLC. Panel A: ,
FGLM, 1:8,000; , FSWGAEGQR, 1:1,000. Panel B: , LPPSR,
1:2,000; , ASTTTNYT, 1:1,000
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Non-pNA bioactive peptides of 8-28 amino acids were
excellent substrates at E:S molar ratios of 1:400 to 1:600
(Table III). Angiotensins I and II were cleaved relatively rapidly
(Fig. 3) with complete hydrolysis by 50 and 100 min, respectively, at these very low ratios. VIP was fragmented
somewhat more slowly, 40% being cleaved by 90 min; atrial natriuretic
peptide, bradykinin, substance P, and neurotensin were only slowly
degraded. These results indicate that peptidase IImes also
has oligopeptidase activity. This is not a novel concept because
multiple reports indicate that many purified enzymes have both
aminopeptidase and oligopeptidase activity. These include cathepsin H
(20). In addition, many peptidyldipeptidases, including cathepsin B
(21), also have oligopeptidase activity. In all cases, this has been demonstrated with peptide substrates rather than with proteins, a
result that is paralleled in this study.

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Fig. 3.
Decrease in percent of peak area of various
bioactive peptides from HPLC after incubation with mesquite pollen
peptidase IImes. Purified enzyme was incubated with
angiotensins I and II, VIP, atrial natriuretic peptide, bradykinin,
substance P, or neurotensin in 0.1 M Tris-HCl, pH 8.0, at
37 °C as described under "Methods." At various times aliquots
were removed, acidified to stop the reaction, and peaks were separated
on HPLC. , angiotensin I, 1:480; , angiotensin II, 1:600; ,
VIP, 1:570; , atrial natriuretic peptide, 1:450; , bradykinin,
1:510; , substance P, 1:400; , neurotensin, 1:500.
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The hydrolysis of all bioactive peptides occurred exclusively and
internally after isoleucine, leucine, phenylalanine, alanine, and
methionine residues (most rapidly after isoleucine) in the substrates
tested but not after every such residue in every peptide. Six of the
peptides had one to three cleavages, but VIP was cleaved at seven
sites. Hydrolysis after methionine residues also occurred in the
dipeptides Met-Phe and Met-Tyr, which are usually used as internal
standards in determining kinetic constants by HPLC. Also, a small
amount of inhibition (20%) of the hydrolysis of the bioactive peptides
occurred when Met-Phe or Met-Tyr were present. No preference for either
hydrophilic or hydrophobic residues in either the P1' or
P2 position was obvious. (The amino acid residues in
substrates are numbered as P3, P2,
P1, etc. toward the NH2 terminus from the
cleavage site and P1', P2', P3',
etc. toward the COOH terminus (22).)
Although phenylalanine or alanine was the favored
NH2-terminal residue for aminopeptidase activity and
isoleucine for the oligopeptidase activity, cleavage was exhibited in
both cases after all three residues. Significantly, no
NH2-terminal amino acid cleavage occurred with the
bioactive peptides tested because none had a suitable hydrophobic
residue in that position, supporting our contention of a single enzyme
with two activities of defined specificities.
As with mesquite pollen peptidase Imes, peptidase
IImes only very slowly hydrolyzed proteins, such as
azocasein and blue hide powder (a matter of days), whereas the plasma
serpins,
1-proteinase inhibitor and
1-antichymotrypsin, were not hydrolyzed despite the
known susceptibility to proteolytic attack within their respective reactive site loops. These results differ from data obtained recently with a chymotrypsin-like peptidase from ragweed pollen which rapidly inactivated human
1-proteinase inhibitor (15).
Inhibition Profile--
Peptidase IImes was not
inhibited by cysteine or metalloproteinase inhibitors (Table
IV) or by the specific serpins
1-proteinase inhibitor and
1-antichymotrypsin. It was, however, inactivated by low
Mr serine proteinase inhibitors such as
diisopropyl fluorophosphate, AEBSF, and 3,4-dichloroisocoumarin,
although at rather high concentrations. As expected, TPCK was a good
inhibitor, by virtue of the specificity of the enzyme toward
phenylalanine residues, whereas TLCK was not inhibitory. In support of
the exopeptidase activity exhibited by peptidase IImes, the
aminopeptidase inhibitor bestatin was very effective in reducing enzyme
activity on synthetic substrates. However, both 1,10-phenanthroline (a
metal chelator) and 4,7-phenanthroline (a non-chelator) inhibited as
well. The non-chelating analog can bind nonspecifically to the active
site of some enzymes (23), which indicates that no metal ion is
involved in the enzymatic activity.
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Table IV
Effect of class-specific inhibitors on the amidolytic activity of
mesquite pollen peptidase IImes
Results are for a 15-min incubation at 25 °C in 0.1 M
Tris-HCl, pH 8.0, with 1 mM H-Ala-pNA as
substrate.
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In support of the specificity of peptidase IImes toward
substrates with P1 hydrophobic residues, a series of
chloromethyl ketones and phosphonates was effective in blocking
amidolytic activity with varying success (Table
V). Significantly, the best inhibitory
activity was found with an unblocked chloromethyl ketone. The
concentrations of these inhibitors were much lower than the concentration of the substrate H-Ala-pNA (1:10 and lower
molar ratios), indicating that this was not a competitive inhibition, in contrast to results obtained with NH2-terminal blocked
substrates, as discussed above.
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Table V
Effect of peptide inhibitors on the amidolytic activity of mesquite
pollen peptidase IImes
Results are for a 15-min incubation at 25 °C in 0.1 M
Tris-HCl, pH 8.0, 0.15% Me2SO with 1 mM
H-Ala-pNA as substrate.
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Enzyme Kinetics--
The kinetic activity parameters of mesquite
pollen peptidases Imes and IImes on a variety
of substrates are set forth in Table VI.
H-Ala-pNA and H-Ala-Ala-pNA were again the
substrates most preferred by peptidase IImes.
H-Leu-pNA appeared to be a better substrate than it did in
Table II where activity was displayed essentially as
kcat and for H-Ala-pNA was 40 times
greater than for H-Leu-pNA. However, the
Km of H-Leu-pNA was 20 times smaller than
H-Ala-pNA and thus had a
kcat/Km only slightly lower than the
latter substrate. These results essentially parallel data shown earlier
which were utilized in determining enzyme specificity (Table II). It
should be noted that in the bioactive peptides analyzed, particularly
angiotensins I and II, hydrolysis of the isoleucine-histidine peptide
bond occurred five to six times faster than for H-Ile-pNA.
The increased rate was apparently the result of greater affinity of the
enzyme for these peptide substrates because the
kcat values were nearly the same as for
H-Ile-pNA, whereas the Km was six to
eight times lower.
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Table VI
Kinetic parameters of mesquite pollen peptidases Imes and
IImes with peptide substrates and comparison to other enzymes
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Peptidase Imes also cleaved both angiotensin II and atrial
natriuretic peptide rapidly (complete hydrolysis in 60 and 90 min at
1:7,000 and 1:3,000 molar ratios, respectively (14)) but, as described
previously, after an arginine residue. The
kcat/Km values were similar
to those found for peptidase IImes, also as shown in Table
VI.
Internal Sequence Comparison with That of Known
Proteins--
Because the NH2 terminus of peptidase
IImes was blocked, making it impossible to perform a
comparison with the structures of other possibly related peptidases,
the enzyme was cleaved internally with P. gingivalis
Arg-gingipain, a cysteine proteinase that hydrolyzes after arginine
residues (19). Although several peptide fragments were found, one
peptide specifically was obtained which had an NH2-terminal
KITFYQDRPDIMARYTLKIEADKYLYPVELSN. Significantly, this structure had
a 64% homology with the zinc-containing aminopeptidase N (membrane
alanine aminopeptidase) from Escherichia coli and 59%
homology with aminopeptidase N from Haemophilus influenzae. The combined inhibitory activities of both 1,10- and 4,7-phenanthroline indicated the absence of a metal; however, the sequence found corresponded to residues 127-157 in aminopeptidase N from E. coli, whereas the zinc ion ligands in that enzyme were at residues
296, 300, and 319. Thus, this sequence was far from any of the zinc binding sites and may act like a mosaic protein as exemplified by the
S8 serine peptidase from Vibrio alginolyticus (24), a member
of the subtilisin family which acts as an endopeptidase with homologous
domains similar to those found in metallopeptidases, including an
aminopeptidase from Vibrio proteolyticus (family M28) and an
endopeptidase of the thermolysin family (M4).
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DISCUSSION |
Pollen is one of the well known triggers of bronchial
hyperresponsiveness, or exaggeration of response to inflammation,
observed in allergic asthma. Although mesquite does not have a
widespread distribution, it has recently been cultivated extensively in
the southcentral and southwestern United States, thereby increasing its
contact with people and making its pollen a serious spring aeroallergen
(25). Once the pollen grains come in contact with an aqueous
environment, such as the mucus layer in the lung airways, they swell
and split and release many proteins (26). Pollen proteins that are
allergens and elicit an immunological response have been studied
abundantly (27-31). However, some of these allergens in fact appear to
have, in addition, enzymatic functions displaying lyase (32), esterase
(33), and polygalacturonase (34) activities. Indeed, some dust mite
allergens, such as Der p I and Der f I (from
Dermatophagoides pteronyssinus and Dermatophagoides
farinae, respectively), are cysteine proteinases, and Der p
III and Der f III are serine proteinases (35).
Both mesquite and ragweed pollens have yielded peptidases with both
trypsin-like and chymotrypsin-like specificities (14-16). This report
concerns the results obtained in the study of a second mesquite pollen
activity (peptidase IImes) that was quite different from
the others. The enzyme manifested both aminopeptidase and oligopeptidase activity, based on results with blocked and unblocked peptide pNAs and with unblocked polypeptides. The data
obtained suggested the importance of hydrophobic residues for both
activities, with phenylalanine being preferred for aminopeptidase
function and multiple hydrophobic residues required in the
P1 position for internal cleavage. Such a combination of
activities is not unusual and has been observed for cathepsin H (20).
In addition, cathepsin B has been shown to have both
peptidyldipeptidase and oligopeptidase activities (21).
It is important to note that homology with aminopeptidases from other
organisms was obtained readily in analysis of a single peptide fragment
from peptidase IImes. The complete amino acid sequence of
peptidase Imes was shown previously (14) to be homologous with protease II from E. coli, a member of the
prolylendopeptidase family. Recent results comparing structures of a
trypsin-like oligopeptidase isolated from suspension-cultured soybean
cells found that homology also existed between that peptidase and
prolylendopeptidases (human or porcine) including protease II (E. coli) (36). In fact, the soybean oligopeptidase resembled
peptidase Imes in other ways as well; cleavage after
arginine and, to a lesser extent, lysine residues, hydrolysis of
peptides only, a serine peptidase specificity, and molecular mass of 90 kDa. Whether homologies exist between peptidase IImes and
endopeptidases from other organisms remains to be established, but
is likely.
The rapid hydrolysis of the bioactive peptides VIP and
angiotensins I and II may be of potentially physiological significance. By rapidly degrading and inactivating VIP (a bronchodilator) while only
slowly hydrolyzing substance P (a bronchoconstrictor), the peptidase could be expected to exacerbate the overall
bronchoconstrictive effect detected in asthmatic lungs. In addition,
angiotensin II is a potent vasoconstrictor (13), and its cleavage and
inactivation by the peptidase could also be expected to contribute to
the overall vasodilation observed in asthmatic lungs. Kinetic rate
constants indicate that the rate of cleavage of two of these peptides
(angiotensins I and II) in vitro was relatively rapid and
comparable to the rates of hydrolysis of peptide bonds by other well
known proteinases, such as chymotrypsin (37).
However, the fragmenting of both angiotensins I and II into smaller
peptides could have important effects of their own. Macrophages appear
rapidly in the lung after local allergen challenge, suggesting a rapid
migration of monocytes (38). The two tetrapeptides (DRVY and IHPF) of
angiotensin II are chemotactic factors for neutrophils and,
particularly, monocytes (39), which exhibit 50% of the optimal
response to C5a (a potent chemotactic factor) at very low
concentrations of the peptides. The cleavage of angiotensin II by
peptidase IImes produced a pentapeptide (DRVYI) and a
tripeptide (HPF). Because the former peptide contains the chemotactic
NH2-terminal tetrapeptide it could be an effective
chemotactic factor as well.
Many of the proteins extracted from pollen are enzymes that are no
doubt normally involved in the germination of the plants (40). Under
abnormal conditions, however, such as allergic asthma triggered by
pollen, these enzymes may play a role in the pathology of the disease.
Because the mesquite pollen peptidase IImes with both exo-
and oligopeptidase specificity described here degrades both VIP and
angiotensins I and II, this enzyme may have the potential for making a
significant contribution to the pathological effects observed in
allergy-related asthma.