From the
The existence of a protein
The granule-associated mast cell and leukocyte serine proteases
vary strikingly in specificity and function
(1) . A subset of
these enzymes, including mast cell tryptases, are tryptic in
specificity, hydrolyzing target peptides on the COOH-terminal side of
basic residues (2, 3). Tryptases, the expression of which is confined
almost exclusively to mast cells in human tissues
(4) , are the
secretory granule's major
protein
(5, 6, 7) . They may regulate vascular
and bronchomotor tone, act as local anticoagulants, and influence
tissue remodeling in response to inflammation by activating matrix
metalloproteinases and promoting the growth of
fibroblasts
(8, 9) . In humans and mice, there are two or
more closely related tryptase genes (10-14). The existence of
additional more distant relatives of mast cell tryptases was suggested
by the serendipitous cloning from dog mastocytoma cells of a cDNA whose
sequence predicted a protein with certain similarities to known
tryptases
(15) . RNA blots and immunohistochemical studies using
antibodies raised against a portion of the predicted sequence suggested
that the gene encoding this protein, termed dog mast cell protease-3
(dMCP-3),
Column fractions also were assayed for dMCP-3
immunoreactivity by probing dot blots with
anti-dMCP-3
This report describes the purification and properties of an
oligomeric, trypsin-like protease isolated from dog mastocytoma cells.
The findings establish that the protein predicted from a previously
sequenced ``orphan'' cDNA cloned from a dog mastocytoma
library
(15) is translated and processed into a catalytically
active serine protease, dMCP-3, which is abundantly expressed in the G
line of dog mastocytomas. RNA blotting and immunohistochemical studies
suggest that a similar or identical enzyme is present in normal dog
mast cells
(16) . Several features ally dMCP-3 with the group of
known mast cell tryptases, although there are potentially key
differences, such as stabilization by heparin, formation of
intersubunit disulfide bonds, substrate preferences, and inhibitor
susceptibility.
dMCP-3's gel filtration behavior suggests an
oligomeric structure in the native, active state. The M
Subunit
association in dMCP-3, as in dog and human tryptase, may be sustained
in part by noncovalent hydrophobic interactions. Tryptase tetramers
derive additional stability through largely electrostatic interactions
with heparin and other sulfated proteoglycans
(17, 31) ,
but do not form covalent attachments between subunits and do not
contain free sulfhydryl groups in the nonreduced state
(32) . The
dMCP-3 oligomer, being stable in heparin's absence, may be
supplementally stabilized by the formation of disulfide linkages
between some subunits. The cDNA-derived amino acid sequence of dMCP-3
predicts the presence of 4 more cysteines (Cys
The predicted net charge of
dMCP-3 ((Arg + Lys) - (Asp + Glu)) is -11,
compared to dog tryptase, which is -3
(15) . Both enzymes
contrast strikingly with dog chymase (predicted net charge of
+16). Despite its strong predicted negative charge, dMCP-3 binds
to polyanionic heparin (albeit less strongly than does tryptase), as
reflected by its binding to a heparin affinity column. 5 units of the
net charge difference between dMCP-3 and tryptase are due to a
disparity in the number of positively charged residues (13 versus 18). The remaining 3 units of difference are due to a disparity in
negatively charged residues (24 versus 21). Thus, the ability
of oligomeric tryptase-like serine proteases to bind to heparin appears
to be conferred by a relatively small number of critically positioned
basic residues. This contrasts with cationic chymases and human
cathepsin G (net charge +22), whose ability to bind to heparin is
thought to be a function of a considerably larger number of cationic
amino acid side chains concentrated in two large surface
patches
(34, 35, 36) .
The results of the
peptidyl pNA studies establish differences in substrate preferences
between dMCP-3 and dog tryptase. The difference in the rate of
hydrolysis of VGRpNA is particularly striking. It is reasonable to
hypothesize from the contrasting rates of synthetic substrate
hydrolysis that physiological targets of dMCP-3 and tryptase differ and
that the two enzymes serve different functions. In the case of
tryptases, several natural peptide and protein targets have been
identified in vitro, although the importance of these targets
awaits validation in vivo. In the case of dMCP-3, one putative
physiological substrate identified in the current study is calcitonin
gene-related peptide. Several studies suggest that the flare reaction
caused by this neuropeptide in the setting of cutaneous neurogenic
inflammation is limited by mast cell
proteases
(23, 37, 38) . dMCP-3 may be among
these proteases. On the other hand, in contrast to tryptases, dMCP-3
does not degrade vasoactive intestinal peptide and will not directly
influence bronchodilation and other consequences of this
peptide's neural release. This finding, and the lack of
caseinolytic activity, predict that dMCP-3 is highly selective for its
targets. Identification of additional natural targets requires more
investigation.
Like mast cell tryptases, dMCP-3 resists inactivation
by large, natural inhibitors of most trypsin-like serine proteases. In
tryptases, this idiosyncrasy is poorly understood in molecular terms.
The major distinction between dMCP-3 and dog tryptase in inhibitor
susceptibility is the former's resistance to inhibition by
aprotinin. In this respect, dMCP-3 is similar to human
tryptases
(6, 30) , even though dog tryptase is more
closely related to human tryptases in primary structure than dMCP-3 is
to any known tryptase. Nonetheless, dMCP-3 and tryptases alike are
inactivated by several low molecular weight trypsin-like serine
protease inhibitors, which, because of their small size, do not make
extensive contacts with amino acid residues in the vicinity of the
substrate binding cleft. An especially effective dMCP-3 inhibitor is
BABIM
(24) , which, by analogy to benzamidine (a related aromatic
amidine), is thought to reversibly inactivate dMCP-3, tryptases, and
trypsin by occupying the portion of the active site that accommodates
the substrate P1 lysine or arginine side chain, whose protonated amino
group forms a salt bridge with the carboxyl anion of an internal
residue (Asp
The technical assistance of Jeanne Aufderheide and
Lianjie Du in the purification of lung tryptase and of Barbara A. Kirby
in the peptide hydrolysis experiments is greatly appreciated.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
48% identical with mast cell
tryptases was predicted previously from a dog mastocytoma cDNA.
Antibodies raised against a peptide based on the deduced sequence
suggested that the protein (dog mast cell protease-3, dMCP-3) is
expressed in mast cells. In this report, characterization of the
protein purified from mastocytomas reveals an N-glycosylated,
high molecular weight, tryptic serine protease, which appears to be a
tetramer of catalytic subunits, approximately half of which are linked
by disulfide bonds. The oligomeric complex yields a single
NH
-terminal sequence, which is identical with that
predicted by dMCP-3 cDNA. This finding, and the lack of closely related
genes on blots of genomic DNA, predict that each subunit is the product
of one gene. Although dMCP-3 binds to heparin, it is active and stable
at low ionic strength in heparin's absence. It resists
inactivation by inhibitors in plasma but is sensitive to small
inhibitors, e.g. leupeptin and
bis(5-amidino-2-benzimidazolyl)methane (BABIM). dMCP-3 hydrolyzes
extended peptidyl p-nitroanilides ending in basic residues,
with P1 arginine preferred to lysine; it hydrolyzes the
Arg
-Ser
bond of calcitonin gene-related
peptide but cleaves neither vasoactive intestinal peptide nor casein.
These data suggest that dMCP-3 is a unique serine protease whose
stability, formation of intersubunit disulfide bonds, inhibitor
susceptibilities and substrate preferences differ from those of its
closest relatives, the mast cell tryptases.
(
)
is transcribed and translated in dog
mastocytomas, normal dog tissue mast cells, and possibly
neutrophils
(16) . Immunoblotting of electrophoresed cell
extracts confirmed the presence of immunoreactive material of
approximately the expected size (
30 kDa) in dog
mastocytomas
(16) . The work below describes the purification and
characterization of dMCP-3, a novel member of the family of tryptic
mast cell proteases.
Enzyme Assays
Aliquots from purification steps
were tested for amidolytic activity with 0.135 mMN-benzoyl-L-Val-Gly-Arg-p-nitroanilide
(VGRpNA) in 60 mM Tris (pH 7.8) with 50 µg/ml bovine lung
heparin. VGRpNA was dissolved in dimethyl sulfoxide to 20 mg/ml, then
diluted in assay buffer. Release of p-nitroaniline at 37
°C was detected by monitoring change in A
as described
(17) ; moles of substrate hydrolyzed were
calculated using an
of 11,100
M
cm
. For convenience,
column fractions were routinely monitored for
N
-benzyloxycarbonyl-L-Lys-thiobenzylester-hydrolyzing
activity using modifications of a microplate assay
(18) . For
this assay, 50-µl aliquots of column fractions were mixed with 100
µl of 1 mM 5,5`-dithiobis(2-nitrobenzoic acid) (DTNB) in a
solution of 10 mM Hepes (pH 7.2) containing 1 mM
CaCl
and 1 mM MgCl
in single wells of
U-Bottom Microtest III 96-well assay plates (Becton Dickinson). 50
µl of 2 mM substrate in assay buffer were added to the
wells. A
at 37 °C was measured on a
Thermo
microplate reader (Molecular Devices, Menlo Park,
CA). Protein concentration in samples was determined by Bradford assay
(Bio-Rad), using BSA as a standard, or by measuring the
A
of purified proteins. The
of dMCP-3 was calculated to be 74,380 M
cm
based on tyrosine and tryptophan content
deduced from dMCP-3 cDNA
(15) .
Purification
Tumors of the BR and G dog
mastocytoma lines propagated in athymic mice as described
(19, 20) were harvested and frozen at -70 °C until
extraction. Thawed tumors were washed in a solution of 150 mM
NaCl in 10 mM Tris (pH 7.5), then homogenized in 10 ml of 10
mM Bis-tris (pH 6.1) per g of tissue. The supernatant
generated by centrifuging the homogenate for 30 min at 27,500
g was brought to 20% glycerol and 1.6 M NaCl and
applied to a 10-ml column of benzamidine-Sepharose 6B (Pharmacia
Biotech) equilibrated with a solution of 8 mM Bis-tris (pH
6.1), 20% glycerol, and 1.6 M NaCl. After loading with
supernatant, the column was washed with equilibration buffer and eluted
with 30 ml of 0.15 M benzamidine in the same buffer. To remove
NaCl and benzamidine, the column eluate was passed in 10-ml aliquots
through 50 ml of Sephadex G-25 (Pharmacia) equilibrated with 20%
glycerol in 8 mM Bis-tris.
3-ml aliquots of G-25 eluate,
each containing
450 µg of protein, were applied to a 7.5
75 mm heparin-5PW high pressure liquid chromatography column
(Toso-Haas, Montgomeryville, PA) equilibrated with 20% glycerol in 8
mM Bis-tris (pH 6.1). Fractions eluted with a gradient of 0 to
1.6 M NaCl in the glycerol/Bis-tris buffer were collected,
assayed for tryptic esterase activity as above, and analyzed by
SDS-polyacrylamide gel electrophoresis (PAGE) on 12.5% gels stained
with Coomassie Blue.
, a rabbit polyclonal antiserum
raised against a synthetic peptide corresponding to residues
166-181 of catalytic domain sequence deduced from dMCP-3
cDNA
(15, 16) . Aliquots of samples were transferred to
prewetted nitrocellulose. Blots were blocked by incubation for 1 h with
a solution of 1% BSA and 150 mM NaCl in 10 mM Tris
(pH 7.5), then incubated for 1 h with a 1:1000 dilution of
anti-dMCP-3
antiserum in a solution of 10
mM Tris (pH 7.5) containing 150 mM NaCl and 0.3%
Tween 20. After washing of blots with detergent buffer alone,
immunoreactive material was detected with a horseradish
peroxidase-labeled goat anti-rabbit IgG (1:1000; Bio-Rad) incubated
with 0.5 mg/ml 4-chloro-1-naphthol in 0.15% H
O
.
NH
An
8-µg sample of purified dMCP-3 was subjected to automated
NH-terminal Sequencing
-terminal sequence analysis on an Applied Biosystems
gas-phase sequenator. The analysis was carried out by the University of
California, San Francisco's Biomolecular Resource Center.
Thiol Titration
Free thiols were assayed using
DTNB as described
(21) . In brief, 0.01 ml of a solution of 4
mg/ml DTNB in 1% SDS, 40 mM NaPO
(pH
8.0) was added to 0.5 ml of the same buffer containing dMCP-3, BSA, or
carbonic anydrase. After 15 min, A
was
measured. The assay mixture was referenced against a mixture of buffer
plus DTNB alone.
Deglycosylation
Purified dMCP-3 was deglycosylated
with peptide N-glycosidase F (Genzyme). After boiling 2 µg
of DMCP-3 for 5 min in the presence of 0.5% SDS and 0.3 M
2-mercaptoethanol, the mixture was diluted 3-fold with a solution
containing 100 mM NaHPO4 (pH 7.5), 10 mM
EDTA, and 1% Nonidet P-40 and was incubated for 16 h at 37 °C with
0.3 unit of glycosidase. A control aliquot of dMCP-3 was subjected to
the same treatment in the absence of glycosidase. The reaction was
stopped by the addition of an equal volume of SDS-PAGE sample buffer
(4% SDS, 20% glycerol, 10% 2-mercaptoethanol in 0.125 M
Tris-Cl (pH 6.8)), and the products were analyzed by SDS-PAGE.
Size Exclusion Chromatography
Purified dMCP-3 was
injected onto a TSK-250 Bio-Sil column equilibrated with a solution of
10 mM Tris-Cl (pH 7.4), 2 M NaCl, and 20% glycerol.
The elution volume of dMCP-3, as identified by peak esterase activity
and A of the eluate, was compared to that of
dog tryptase, cyanocobalamin, and globular protein standards eluted
with the same solution. The M
of dMCP-3 was
estimated by extrapolation from a plot of molecular standard
K
versus log M
.
Stability of Amidolytic Activity
To compare the
stability of dog tryptase and dMCP-3, purified preparations of each
enzyme were incubated separately for 1 h in the presence or absence of
bovine lung heparin (50 µg/ml). Incubations were carried out at 37
°C in polypropylene tubes containing 10 mM Tris-Cl (pH
7.4) and 150 mM NaCl. Aliquots of incubating enzyme solutions
were withdrawn at intervals to assay residual 0.1 mMN-p-tosyl-Gly-L-Pro-Arg-pNA
(GPRpNA)-hydrolyzing activity.
Substrate Specificity
To compare the substrate
preferences of dMCP-3 and dog tryptase, the activity of the two enzymes
was tested in parallel against a battery of peptidyl pNAs. Substrates
were diluted from 20 mg/ml stocks in dimethyl sulfoxide to 0.2
mM in 60 mM Tris (pH 7.8) containing 50 µg/ml
heparin. 30 ng of purified dog tryptase or dMCP-3 were mixed with
substrate-containing assay buffer to a final volume of 200 µl in
single wells of 96-well assay plates. Substrate hydrolysis at 37 °C
was monitored at 405 nm in a microplate reader as described above.
Assays were performed in duplicate in three separate experiments. All
substrates were obtained from Sigma with the exception of
N-benzoyl-L-Lys-Gly-Arg-pNA (KGRpNA),
which was obtained as described
(17) .
Hydrolysis of Azocasein and Bioactive Peptides
To
test the general proteinase activity of dMCP-3, aliquots of dMCP-3,
tryptase, or trypsin stocks were added to a solution of azocasein
(final concentration 14 mg/ml; Sigma) in 60 mM Tris-Cl (pH
7.8). The final concentration of dMCP-3, tryptase, and trypsin in their
respective incubation solutions was 5.0, 3.6, and 5.4 µM,
respectively. Unhydrolyzed azocasein remaining after 10 min of
incubation at 40 °C was precipitated by addition of a 5-fold
greater volume of 5% trichloroacetic acid. The resulting mixture was
centrifuged twice for 5 min at 16000 g to obtain a
supernatant, the A
of which was measured
after zeroing against a solution of azocasein incubated and acidified
in the same manner, without added protease. To test the ability of
dMCP-3 to hydrolyze selected bioactive peptides, dMCP-3 was incubated
in separate experiments with substance P, vasoactive intestinal
peptide, and calcitonin gene-related peptide (Sigma). As controls, the
same peptides were incubated with human lung tryptase (purified as
described previously
(22) , with minor modifications) or with
buffer alone. In each incubation mixture, the concentration of peptide
was 0.1 mM and that of dMCP-3 or tryptase (based on protein
measurement) was 0.1 µM and 0.2 µM,
respectively. As tested in a microplate-based assay using VGRpNA as a
substrate, the specific activity of dMCP-3 used in these experiments
was approximately twice that of the human lung tryptase. Protease
stocks contained 2 µg of bovine lung heparin per µg of dMCP-3
or tryptase. Solutions formed by addition of protease and peptide to
120 mM Tris (pH 7.4) containing 140 mM NaCl were
incubated for 30 min at 37 °C. Aliquots withdrawn at intervals were
injected onto a Vydac C18 reverse phase column (The Separations Group;
Hesperia, CA) and eluted using a linear gradient of 10-40%
acetonitrile in 0.1% trifluoroacetic acid, as described
(23) with monitoring of A
.
Enzyme pH Activity Curve
Activity of purified
dMCP-3 (94 ng/ml) was measured at 37 °C using 0.3 mM
GPRpNA in 100 mM Bis-tris (pH 5.5-6.5) or Tris-Cl (pH
7.0-9.0).
Inhibitor Profile
Purified dMCP-3 (3 µg/ml;
100 nM) was preincubated in 60 mM Tris (pH 7.8)
with 50 µg/ml bovine lung heparin for 10 min at 37 °C with each
of a panel of inhibitors. Activity remaining after preincubation was
determined in an assay for amidolytic activity initiated by the
addition of VGRpNA as described above. In control assays, dMCP-3 was
preincubated under identical conditions in the absence of inhibitor. To
improve solubility, some inhibitors were incubated with dMCP-3 and
assay buffer in the presence of 1% dimethyl sulfoxide or
dimethylformamide. Pilot studies established that neither solvent at
this concentration alters dMCP-3 amidolytic activity. All inhibitors
were obtained from Sigma except for leupeptin (Boehringer Mannheim),
BABIM (from R. Tidwell
(24) ), secretory leukocyte protease
inhibitor (from J. Kramps), and dog plasma, which was prepared from
heparinized blood.
Genomic DNA Blotting
Genomic DNA was purified from
dog and human circulating mononuclear cells, which were purified in
each case from 10 ml of fresh blood. Blood diluted with 12 ml of
Ca- and Mg
-free Hank's
balanced salt solution was overlaid with 15 ml of Histopaque-1077
(Sigma) and centrifuged for 30 min at 400
g. Cells
from the mononuclear band were lysed and digested in 10 mM
Tris-Cl (pH 8) containing 100 mM NaCl, 25 mM EDTA,
0.5% SDS, and 0.1 mg/ml proteinase K. The resulting suspension was
shaken for 12-18 h at 50 °C. DNA was isolated by
phenol-chloroform extractions followed by ethanol precipitation. After
digestion with EcoRI or HindIII, the resulting dog
and human DNA was electrophoresed in 1% agarose and transferred to a
nylon membrane (Nytran Plus; Schleicher and Schuell). The membrane was
washed for 5 min in 75 mM sodium citrate (pH 7.0) containing
0.75 M NaCl, air-dried, baked for 2 h in a vacuum oven at 80
°C, then prehybridized in 90 mM sodium citrate (pH 7.0)
containing 50% formamide, 0.9 M NaCl, 0.5% SDS, 1 g/liter each
of Ficoll 400, polyvinylpyrrolidone, and BSA, and 100 µg/ml salmon
sperm DNA. Hybridization was initiated by the addition of a
P-labeled cDNA probe to the above solution. The probe was
generated by random priming of previously cloned and sequenced
1174-base pair dMCP-3 cDNA
(15) (excised from pBluescript using
EcoRI) to a specific activity of
2
10
cpm/µg (using [
-
P]dCTP,
[
-
P]dGTP, and a Megaprime kit, Amersham).
After 15 h of hybridization, the membrane was subjected to washes of
escalating stringency, followed by autoradiography.
Purification
The results of a single,
representative set of chromatographic steps leading to the purification
of 7.1 mg of dMCP-3 from G mastocytoma extract are summarized in
. The benzamidine-Sepharose step effected 62-fold
purification of VGRpNA-hydrolyzing activity from the crude, low ionic
strength extract. The VGRpNA-hydrolyzing activity of the
benzamidine-Sepharose eluate is due to the presence of both dMCP-3 and
tryptase, which are separated in the heparin affinity step. The -fold
purification of dMCP-3 falls slightly from the benzamidine-Sepharose to
the heparin affinity step because of the removal of tryptase, whose
specific activity toward VGRpNA is higher than that of dMCP-3 (see
below). As shown in Fig. 1, heparin affinity chromatography
reveals two widely separated peaks of A, the
first of which is due to dMCP-3 and the second of which is due to
tryptase. Heparin-column fractions spanning these two peaks are the
only fractions with VGRpNA-hydrolyzing activity. Neither
A
nor amidolytic activity was detected in
flow-through fractions. Fig. 1also suggests that the
concentration of dMCP-3 is higher in G than in BR mastocytoma extracts
and demonstrates a difference between BR and G mastocytomas in relative
content of dMCP-3 and tryptase. Although dMCP-3 is the principal
VGRpNA-hydrolyzing enzyme in low ionic strength extracts of both
mastocytoma lines, only BR extracts contain substantial tryptase. Prior
work in this laboratory demonstrated that most tryptase and most
tryptic amidolytic activity in BR homogenates is soluble only when
extracted using buffer of high ionic strength
(17, 25) .
High ionic strength extracts of G cells, on the other hand, contain
little tryptic activity
(25) and negligible dMCP-3 (not shown).
Therefore, dMCP-3 is the major tryptic protease of G mastocytomas. BR
mastocytomas contain substantial amounts of both dMCP-3 and tryptase,
with most of the former extracting into low ionic strength buffer and
most of the latter into high ionic strength buffer.
Figure 1:
Heparin affinity chromatography of
mastocytoma tryptase and dog mast cell protease-3 (dMCP-3). Tryptic
activity extracted from mastocytoma cell homogenates was purified
initially on benzamidine-Sepharose, loaded onto a heparin-5PW column,
and eluted with a gradient of NaCl (as shown by the dotted
lines) to separate dMCP-3 from tryptase. A and B show the A of protein derived from low
ionic strength extracts of ``BR'' and ``G''
mastocytoma cells, respectively. Tryptic activity (not shown) in column
fractions corresponded to the peaks of A
. As
reflected in the relative peak sizes in the chromatograms in the two
panels, dMCP-3 is by far the major tryptic protease in cell extracts
and benzamidine-Sepharose eluates derived from G
mastocytomas.
NH
dMCP-3
corresponding to the ``dMCP-3'' peak of Fig. 1A yielded the following sequence: IVGG()KVPARRY, which corresponds
exactly to the sequence of the catalytic domain (after removal of
signal and activation peptide) deduced from nucleotide sequence of
cloned dMCP-3 cDNA
(15) . The fifth cycle was blank, which is
consistent with modification of the cysteine predicted to occupy this
position.
-terminal Sequence
Free Thiols
An 8 µM solution of
purified dMCP-3 (molarity based on moles of monomer) yielded 9.3
µM DTNB-titratable ``free'' thiol, an average of
1.2 thiols per monomer. Under the same assay conditions, bovine
carbonic anhydrase (which lacks cysteines), yielded 0.05 mol of thiols
per molecule; BSA, with 17 disulfide linkages and 1 unpaired cysteine,
yielded 0.5 thiol per molecule.
Electrophoretic Behavior
The results of SDS-PAGE
of proteins purified from mastocytoma extracts are seen in
Fig. 2
, which emphasizes the dramatic increase in purity achieved
in the benzamidine-Sepharose step. The modest further increase in
homogeneity achieved by the application of the heparin affinity step to
the G mastocytoma material is due to the removal of contaminating
tryptase. A comparison of BR mastocytoma-derived material prepared from
the two heparin affinity peaks shown in Fig. 1A demonstrates a clear difference in electrophoretic behavior
between dMCP-3 and tryptase, with the former migrating as a broad band
centered around 37 kDa. The gel in Fig. 3illustrates the
difference between reduced and unreduced dMCP-3. In the absence of
2-mercaptoethanol, purified dMCP-3 separates into two bands, one
approximately twice the apparent M
of the other,
consistent with the higher band being a disulfide-linked dimer. As is
often the case of proteins containing intramolecular Cys-Cys linkages,
the fully reduced monomer migrates at a higher apparent
M
than that of the unreduced monomer.
Figure 2:
SDS-PAGE of mastocytoma proteins. Samples
were electrophoresed in a gel containing 12.5% acrylamide. All samples
were reduced with 2-mercaptoethanol. Lanes 1 and 7 (MW) contain marker proteins, whose size (in kDa) are
indicated to the left of the gel. These proteins are
phosphorylase B (97.4), BSA (66.2), ovalbumin (42.7), carbonic
anhydrase (31.0), and soybean trypsin inhibitor (21.5). Lane 2 (G LSE) contains 20 µg of protein from crude low salt
extract of G mastocytomas, the starting material for purification of
dMCP-3. Lane 3 (Bz eluate) contains 5 µg of G
mastocytoma-derived protein retained by, and then eluted from,
benzamidine-Sepharose. Lane 4 (dMCP-3) contains 2
µg of protein from the dMCP-3 peak (see Fig. 1) after further
purification by heparin affinity chromatography. Lane 5 (dMCP-3 + PNGase F) contains 2 µg of protein
from the same dMCP-3 peak preincubated with 0.3 unit of peptide
N-glycosidase F to remove asparagine-linked sugars. Lane 6 (PNGase F) contains 0.3 unit of glycosidase alone, which
is a faint band just above that of dMCP-3. Lane 8 (BR
tryptase) contains 2 µg of purified, BR mastocytoma-derived
dog tryptase from the tryptase peak seen in A in Fig. 1.
Lane 9 (BR dMCP-3) contains 2 µg of purified, BR
mastocytoma-derived dMCP-3 from the dMCP-3 peak seen in A in
Fig. 1.
Figure 3:
SDS-PAGE of dMCP-3. Protein contained in
the heparin affinity chromatographic peaks corresponding to dMCP-3, as
identified in Fig. 1, was subjected to 12.5% SDS-PAGE then stained with
Coomassie Blue. Lanes 1 and 2 contain 2.6 µg of G
mastocytoma dMCP-3 prepared for electrophoresis in the absence and
presence, respectively, of 5% 2-mercaptoethanol. The molecular weights
( 10
) and migration positions of marker
proteins (see Fig. 2 legend) are indicated to the left of the
figure.
Post-translational Modification
As seen in
Fig. 2
, incubation of dMCP-3 with N-glycosidase narrows
the protein band and increases mobility. This suggests that most, and
perhaps all, of the heterogeneity of dMCP-3 is due to variable
N-glycosylation at one or both of the consensus sites
identified in the catalytic domain sequence deduced from dMCP-3
cDNA
(15) .
Oligomerization
Protein and esterase activity of
dMCP-3 subjected to analytical TSK-250 size exclusion chromatography
elutes at an apparent M of
196,000, which is
higher than the apparent M
of dog
tryptase
(133, 0) eluted under the same conditions. The
size of the tryptase complex predicted by these experiments agrees with
the value of 132,000 estimated previously by analytical gel filtration
in buffer without glycerol
(17) . In additional experiments, the
elution behavior of dMCP-3 under identical conditions, except for the
presence of 1 mM dithiothreitol, was similar (results not
shown). Pilot gel filtration runs established the need to include
glycerol in the eluting buffer to prevent nonspecific binding of dMCP-3
to the chromatography matrix. 2 M NaCl was included in the
elution buffer to maintain parity of elution conditions with tryptase,
which, if chromatographed in pure form, loses activity rapidly at low
ionic strength. The resulting gel filtration data suggest that native
dMCP-3, like dog tryptase
(25, 26) , is oligomeric, and
that productive interactions between subunits are more likely to be
hydrophobic than electrostatic in nature (Fig. 4).
Figure 4:
Analytical gel filtration of dMCP-3. 20
µl of a 1.5 mg/ml solution of purified dMCP-3 were loaded onto a
Bio-Sil TSK-250 column (7.5 300 mm; Bio-Rad) equilibrated and
eluted with buffer containing 10 mM Tris-Cl (pH 7.4), 2
M NaCl, and 20% glycerol, at a flow rate of 0.5 ml/min.
A
of the eluate was monitored continuously
(A
, solid line), and fractions were
collected for determination of
N
-benzyloxycarbonyl-L-Lys-thiobenzylester-hydrolyzing
activity (Esterase activity,
--
) in
wells of a microtiter plate. The column was calibrated under the same
conditions with molecular standards (M
), including
thyroglobulin (670,000),
-globulin (158,000), ovalbumin (44,000),
horse myoglobin (17,000), and cyanocobalamin (1,350), whose molecular
weights (
10
) and elution positions are
indicated by open arrows. The elution position of dog tryptase
is indicated by the closed arrow. The estimated
M
of the peak of dMCP-3 A
and catalytic activity is
195,000. Esterase activity is the
change in A
/min.
Stability
Comparison of the stability of dMCP-3
and dog tryptase catalytic activity reveals striking differences, as
illustrated in Fig. 5. At low ionic strength, tryptase's
amidolytic activity rapidly declines. After 20 min, <10% of activity
remains. Loss of activity is almost entirely prevented by the inclusion
of heparin in the incubation solution. The activity of dMCP-3, on the
other hand, is stable at low ionic strength in heparin's absence.
In other experiments, dMCP-3 was found to be stable without heparin
after 5 h of incubation at 37 °C, 16 h at 25 °C, or 3 months at
4 °C. SDS-PAGE of dMCP-3 after prolonged incubation fails to reveal
evidence of autodegradation (data not shown). These findings suggest
that purified dMCP-3 is more stable than purified dog tryptase.
Figure 5:
Stability of tryptase versus dMCP-3. Purified dog tryptase (178 nM) and dMCP-3 (175
nM) were incubated separately for 1 h in the presence or
absence of 50 µg/ml heparin. Incubations were carried out at 37
°C in 10 mM Tris-Cl (pH 7.4) containing 150 mM
NaCl. Aliquots of protease solution were withdrawn at intervals during
incubation for immediate assay of residual
tosyl-Gly-L-Pro-Arg-p-nitroanilide-hydrolyzing
activity. Observed residual activity is plotted on a log scale as a
fraction of activity measured at time zero. Tryptase, in the absence of
heparin (--
), loses >90% of catalytic
activity after 10 min of incubation, but is stable when incubated with
heparin (
--
). In contrast, dMCP-3 (closed
squares), is stable in the absence of
heparin.
Preferences for Peptidyl pNAs
Fig. 6
shows a
comparison of peptidyl pNA substrate preferences of dMCP-3 versus those of dog tryptase. The substrate most rapidly hydrolyzed by
dMCP-3 is GPRpNA. The rate of dMCP-3-catalyzed hydrolysis of
N-p-tosyl-Gly-L-Pro-Lys-pNA
(GPKpNA) proceeds at slightly more than half that of GPRpNA, suggesting
a modest preference for P1 arginine over P1 lysine. Both GPRpNA and
GPKpNA are more rapidly cleaved than the other peptidyl pNAs examined,
each of which contains P1 arginine and P2 glycine. Thus, substrates
with P2 proline may be preferred over substrates with P2 glycine. Among
the 3 Xaa-Gly-Arg substrates, KGRpNA is cleaved slightly faster than
N
-benzoyl-L-Ile-Glu-Gly-Arg-pNA,
which is cleaved somewhat faster than VGRpNA, indicating a sensitivity
to variations in the P3 residue, with P3 lysine preferred to P3
glutamic acid, which is preferred to P3 valine. The best of the pNA
substrates for tryptase (i.e. VGRpNA) is among the worst for
dMCP-3. Compared in terms of specific activity, the rate of VGRpNA
hydrolysis per ng of tryptase is
50-fold greater than that per ng
of dMCP-3. Neither dMCP-3 nor tryptase cleaves the standard trypsin
substrate N
-benzoyl-DL-Arg-pNA with
much alacrity, reinforcing the importance of subsite interactions
suggested by the variation in hydrolysis rates among the more extended
pNA substrates. Not shown are results of assays using substrates
lacking a basic residue in the P1 position, including the
chymase/cathepsin G substrates succinyl-L-Val-Pro-Phe-pNA,
succinyl-L-Ala-Ala-Pro-Phe-pNA, and
succinyl-L-Phe-pNA, the elastase substrate
succinyl-L-Ala-Ala-Ala-pNA, and the acidic dipeptide
derivative benzoyl-L-Glu-Glu-pNA. In each case, there is no
detectable hydrolysis by dMCP-3. Thus, dMCP-3 is a peptidyl amidase
with a preference for certain extended substrates with P1 basic
residues. The selectivity for particular tri- and tetrapeptide
substrates with P1 arginine is evidence of an extended substrate
binding pocket.
Figure 6:
Substrate preferences of tryptase
versus dMCP-3. Substrates are
N-p-tosyl-Gly-L-Pro-Arg-p-nitroanilide
(GPR),
N
-p-tosyl-Gly-L-Pro-Lys-p-nitroanilide
(GPK),
N
-benzoyl-L-Ile-Glu-Gly-Arg-p-nitroanilide
(IEGR),
N
-benzoyl-L-Lys-Gly-Arg-p-nitroanilide
(KGR),
N
-benzoyl-L-Val-Gly-Arg-p-nitroanilide
(VGR), and
N
-benzoyl-DL-Arg-p-nitroanilide
(R). All assays were carried out at 37 °C using 0.2
mM substrate in 60 mM Tris-Cl (pH 7.8) containing 50
µg/ml bovine lung heparin. The concentration of each enzyme was 150
ng/ml (
5 nM). The activity unit (
A) is the
observed change in mA/min. Error bars represent standard error
of the mean of 3 independent determinations.
Hydrolysis of Casein
The caseinolytic activity of
dMCP-3 was compared to that of trypsin and dog tryptase in duplicate in
two separate experiments. Although the concentration of dMCP-3 in the
incubation solution was similar to that of trypsin and tryptase, the
rate of hydrolysis of azocasein by dMCP-3 was undetectable, i.e. <0.5% of that of hydrolysis by trypsin. Under these conditions,
however, the rate of azocasein hydrolysis by tryptase was 13% that of
trypsin. Therefore, dMCP-3 possesses minimal general proteinase
activity.
Hydrolysis of Bioactive Peptides
The hydrolysis of
bioactive peptides by dMCP-3 also is limited. Although substance P
contains a potential tryptic hydrolysis site at the
Lys-Pro
bond, it is not hydrolyzed by dMCP-3.
Nor is it cleaved by human lung tryptase, a finding which is consistent
with prior studies
(23, 27) . Vasoactive intestinal
peptide also completely resists degradation by dMCP-3, but is rapidly
hydrolyzed by tryptase, with complete loss of the parent peptide peak
within 5 min of the start of incubation. In contrast, within 30 min,
both dMCP-3 and tryptase hydrolyze all of the calcitonin gene-related
peptide in the incubation solution. dMCP-3 generated just two product
peaks, suggesting a single site of hydrolysis. Amino acid analysis of
the earlier eluting product peak revealed the composition expected of
calcitonin gene-related peptide fragment 19-37 (i.e. 3
Asx, 1 Thr, 2 Ser, 1 Pro, 3 Gly, 1 Ala, 3 Val, 2 Phe, 2 Lys).
Therefore, dMCP-3 hydrolyzes the Arg
-Ser
bond
of calcitonin gene-related peptide. Tryptase, as noted previously,
generates multiple peaks, consistent with two sites of
hydrolysis
(23) . No hydrolysis is detected in solutions of
peptide incubated with buffer alone. Thus, dMCP-3 differs from human
lung tryptase in its ability to hydrolyze bioactive peptides.
pH-Activity Relationship
The amidolytic activity
of dMCP-3 is greatest at alkaline pH. Peak activity plateaus between pH
8 and 8.5 and falls sharply at pH 9 and below pH 7. Activity at pH 5.5,
6.0, 6.5, 7.0, 7.5, and 9.0 is 15%, 21%, 42%, 69%, 92%, and 71%,
respectively, of maximal activity. In the acidic environment of the
mast cell secretory granule, therefore, the activity of dMCP-3 will be
a small fraction of its activity upon secretion into the alkaline
extracellular milieu of the degranulating mast cell.
Inhibitor Susceptibility
As shown in
, dMCP-3 resists inactivation by most serine protease
inhibitors that are proteins, including -proteinase
inhibitor, soybean trypsin inhibitor, ovoinhibitor, and secretory
leukocyte protease inhibitor. It also retains most of its activity
after incubation in 10% dog plasma, suggesting resistance to
anti-proteases in the circulation. The most significant difference
between dMCP-3 and dog tryptase detected in these experiments is in
susceptibility to aprotinin, which dMCP-3 resists and dog tryptase does
not
(17) . Like
tryptases
(6, 17, 24, 28, 29) ,
dMCP-3 is virtually completely inactivated by leupeptin and by BABIM,
and its activity is reduced by high concentrations of NaCl and
CaCl
. Despite the abundance of cysteine residues predicted
by the dMCP-3 cDNA sequence and the evidence of disulfide involvement
in oligomerization, 2 mM dithiothreitol does not diminish
dMCP-3 activity.
Genomic Blots
As shown in Fig. 7,
hybridization of dMCP-3 cDNA to dog genomic DNA reveals strong binding
to one major band in electrophoresed digests of EcoRI- and
HindIII-digested DNA, consistent with the hypothesis that the
subunits constituting the dMCP-3 oligomer are the products of a single
gene. Weaker recognition of one or more bands of similarly digested
human genomic DNA supports the existence of a human homolog.
Figure 7:
Hybridization of a dMCP-3 probe to dog
and human genomic DNA. Restriction digests of dog and human genomic DNA
(10 µg/lane) were electrophoresed in 1% agarose, transferred to a
nylon membrane, hybridized to an 1174-base pair P-labeled
dMCP-3 cDNA probe. The membrane then was subjected to a series of
washes of increasing stringency. The autoradiogram shown was obtained
after washing in 15 mM sodium citrate (pH 7.0), 150
mM NaCl, 0.5% SDS at 65 °C. Lanes 1 and 2 contain EcoRI-restricted dog and human genomic DNA,
respectively. Lanes 3 and 4 contain
HindIII-restricted dog and human DNA, respectively. The size
(in kilobases) and elution positions of HindIII-restricted
marker DNAs are indicated to the left of the figure.
= 196,000 deduced from dMCP-3's elution position on
size exclusion chromatography is nominally consistent with a pentamer
of the M
=
37,000 subunit predicted by
SDS-PAGE. More likely, native dMCP-3 is a tetramer, as dog and human
tryptase appear to be
(6, 17, 25, 30) .
On gel filtration, the larger apparent size of dMCP-3 oligomer may be
due to more extensive attachmentof asparagine-linked glycans, which,
because of extensive hy-dration, occupy more volume in solution per
unit weight than do unmodified globular proteins. dMCP-3 contains two
consensus glycosylation sites, compared to a single site in dog
tryptase. The results of SDS-PAGE also support the concept of dMCP-3 as
an oligomer. Under nonreducing conditions, approximately half of the
purified protein migrates at an apparent M
twice
that of the fully reduced monomer. Thus, approximately half of the
subunits of dMCP-3 form intersubunit disulfide bonds.
,
Cys
, Cys
, and Cys
, according
to numbering in Ref. 15) than are present in mouse and human mast cell
tryptases. Compared to mouse and human tryptases, dog tryptase has one
extra cysteine, which does not correspond to any of the cysteines of
dMCP-3. Mapping of the dMCP-3 sequence onto an existing model of human
tryptase I
(33) predicts that these supernumerary cysteines lie
at or near the subunit surface and that none lies sufficiently close to
another to form an intramolecular Cys-Cys linkage. One or more of these
residues may form linkages between subunits. Of the four cysteines,
Cys
is the least likely to form a disulfide bond because
it is next to a predicted site of N-glycosylation
(Asn
) and because it is the most remote from the
hydrophobic, noncatalytic face of the enzyme thought to be involved in
subunit interactions
(33) . Of the other extra cysteines,
Cys
is the most likely to form an intersubunit disulfide
bond because it appears to lie adjacent to, and between, the
tryptophan- and proline-rich patches, which are conserved in dMCP-3 and
tryptases and are candidate surface sites for contact between subunits
(33). The detection of an average of
1 titratable thiol per
subunit confirms that some of the cysteines of the dMCP-3 oligomer do
not form disulfide bonds. These data, together with the electrophoretic
evidence of subunit dimerization and insights derived from modeling,
suggest that most of the cysteines in the dMCP oligomer are tied up in
intrasubunit Cys-Cys linkages, that some cysteines (one or
more pairs) form intersubunit linkages, and that some are free
thiols. Other cysteines not involved in disulfide linkages may be
modified in some manner (e.g. by oxidation) so that they are
not detected in the free thiol assay.
, using chymotrypsinogen
numbering
(39) ; Asp
of dMCP-3
(15) ).
Another effective inhibitor is leupeptin, a small peptidyl aldehyde,
which in trypsin forms a reversible covalent complex between the
nucleophilic hydroxyl of the catalytically active serine
(Ser
, by chymotrypsin numbering; Ser
of
dMCP-3) and the aldehyde carbonyl (40). This suggests that key elements
of dMCP's catalytic apparatus behave as they do in trypsin.
However, the resistance of dMCP-3 and tryptases to most large, natural
inhibitors of serine proteases suggests that the conformations of their
extended substrate and inhibitor binding site are atypical of
trypsin-like serine proteases. For dMCP-3, an implication of its
exceptional stability and resistance to known antiproteases in plasma
is that it may remain active for extended periods after escape or
secretion from mast cells.
Table:
Inhibition of dog
mast cell protease-3 (dMCP-3)
-benzoyl-L-Val-Gly-Arg-p-nitroanilide;
Bis-tris, bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane; BSA,
bovine serum albumin; PAGE, polyacrylamide gel electrophoresis; GPRpNA,
N
-p-tosyl-Gly-L-Pro-Arg-p-nitroanilide;
KGRpNA,
N
-benzoyl-L-Lys-Gly-Arg-p-nitroanilide;
BABIM, bis(5-amidino-2-benzimidazolyl)methane; GPKpNA,
N
-p-tosyl-Gly-L-Pro-Lys-pNA;
DTNB, 2,2`-dithiobis(2-nitrobenzoic acid).
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.