(Received for publication, March 13, 1995; and in revised form, May 9, 1995)
From the
Mouse mast cell protease 7 (mMCP-7) is a tryptase stored in the secretory granules of mast cells. At the granule pH of 5.5, mMCP-7 is fully active and is bound to heparin-containing serglycin proteoglycans. To understand the interaction of mMCP-7 with heparin inside and outside the mast cell, this tryptase was first studied by comparative protein modeling. The ``pro'' form of mMCP-7 was then expressed in insect cells and studied by site-directed mutagenesis. Although mMCP-7 lacks known linear sequences of amino acids that interact with heparin, the three-dimensional model of mMCP-7 revealed an area on the surface of the folded protein away from the substrate-binding site that exhibits a strong positive electrostatic potential at the acidic pH of the granule. In agreement with this calculation, recombinant pro-mMCP-7 bound to a heparin-affinity column at pH 5.5 and readily dissociated from the column at pH > 6.5. Site-directed mutagenesis confirmed the prediction that the conversion of His residues 8, 68, and 70 in the positively charged region into Glu prevents the binding of pro-mMCP-7 to heparin. Because the binding requires positively charged His residues, native mMCP-7 is able to dissociate from the protease/proteoglycan macromolecular complex when the complex is exocytosed from bone marrow-derived mast cells into a neutral pH environment. Many hematopoietic effector cells store positively charged proteins in granules that contain serglycin proteoglycans. The heparin/mMCP-7 interaction, which depends on the tertiary structure of the tryptase, may be representative of a general control mechanism by which hematopoietic cells maximize storage of properly folded, enzymatically active proteins in their granules.
As much as 30% of the total protein of a mature mouse mast cell
consists of 26- to 36-kDa proteases that are enzymatically active at
neutral pH(1, 2, 3, 4, 5) .
A cDNA that encodes mouse mast cell carboxypeptidase A (mMC-CPA) ()(4) has been cloned, as have the cDNAs and genes
that encode six of the mast cell's seven granule serine
proteases, designated mouse mast cell protease (mMCP) 1 to
mMCP-7(3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13) .
Based on the deduced amino acid sequences of their cDNAs, all mast cell
proteases are initially translated as zymogens that possess a 15- to
19-residue hydrophobic signal peptide and a 2- to 94-residue
``pro'' activation peptide. NH
-terminal amino
acid analyses of the SDS-PAGE-resolved proteins in isolated granule
preparations have revealed that these proteases are preferentially
stored intracellularly in their mature
forms(3, 4, 5) . The function(s) of the
propeptides remain to be elucidated. Although they may be important for
proper folding and/or intracellular targeting of the translated mast
cell serine proteases, most likely the propeptides simply prevent the
proteases from exhibiting enzymatic activity until they are safely
sequestered in the granule. The specific substrates of the mMCPs also
remain to be determined, but mMCP-6 and mMCP-7 have been classified as
tryptases. The other serine proteases have been classified as chymases.
The mature mast cells that reside in the ear and skin of the BALB/c
mouse and the immature mast cells that are derived by culturing BALB/c
mouse bone marrow cells for 2-3 weeks in medium containing
interleukin 3 (mBMMC) express mMCP-7, in addition to mMC-CPA, mMCP-5,
and mMCP-6. BALB/c mBMMC activated at neutral pH through their
FcRI receptors (14) exocytose their
proteases(2) . When the resulting supernatants are
chromatographed on a Sepharose CL-2B column, most mMC-CPA (1) and serine protease (15) activities filter in the
column's excluded volume as a >10
Da complex with
heparin-containing serglycin proteoglycans. Rat (16, 17, 18, 19, 20) and
human (21, 22) mast cell proteases also strongly bind
heparin-containing serglycin proteoglycans. In the case of rat serosal
mast cells, 1 M NaCl or 2 M KCl is required to
separate the components of the macromolecular complex. Thus, even in a
neutral pH environment, much of the macromolecular complex exocytosed
from an activated mast cell remains intact outside the cell.
Johnson
and Barton (13) noted that mast cell tryptases have a higher
content of His than pancreatic trypsin and the other proteases found in
the mast cell granule. While the specific amino acid residues that
interact with heparin-containing serglycin proteoglycans were not
predicted, comparative modeling of a human skin mast cell tryptase
suggested that many of the His residues were on the surface of the
folded human protease, as were Arg and Lys residues. Thus, it was
proposed that the binding of this mast cell tryptase to
heparin-containing serglycin proteoglycans inside the granule might
depend on His residues as well as on Arg and Lys residues. Against this
hypothesis was the observation that human lung mast cell tryptase bound
heparin strongly at neutral pH and that a NaCl concentration >0.8 M was required to dissociate the complex ex
vivo(21) . Moreover, even though BALB/c mBMMC express two
tryptases(6, 11) , 80% of the total serine
protease activity in the pH 7.2 supernatants of Fc
RI-activated
cells resides in the macromolecular complex(15) . Nevertheless,
the observation that mMCP-6 and mMCP-7 are proteins rich in His raised
the possibility that some proteases might dissociate from the
protease-proteoglycan macromolecular complex after the granules are
exocytosed from an activated mouse mast cell into a neutral pH
environment.
To understand how mMCP-7 is packaged in granules and
whether or not this tryptase dissociates from heparin-containing
serglycin proteoglycans outside the mast cell, a comparative modeling
study of mMCP-7 was combined with electrostatic calculations to predict
which residues participate in heparin binding. To test the model, the
pro form of mMCP-7 was expressed in insect cells. Using site-directed
mutagenesis, we confirmed the prediction that His residues 8, 68, and
70 in the properly folded protein are essential for its ionic
interaction with heparin inside and outside the mast cell. We have also
shown that mMCP-7 dissociates from the macromolecular complex
exocytosed at neutral pH from FcRI-activated mBMMC.
His, His
, His
,
His
, and His
in pro-mMCP-7 were each
individually changed to Glu residues with a three-step, site-directed
mutagenesis approach. To make the His
Glu
mutated form of pro-mMCP-7, the first polymerase chain reaction
(PCR) was carried out using a synthetic oligonucleotide
(5`-TTCCGGATTATTCATACCGTCCCACCA-3`) that corresponds to a 5`-region in
pVL1393 and a synthetic oligonucleotide
(5`-GGACAGGAGGCAGAGGGGAACAAGTGG-3`) that spans the desired mutation
site in the mMCP-7 cDNA. A second PCR was carried out using a synthetic
oligonucleotide (5`-CCTTTCCATCCAACGACAAGC-3`) that corresponds to a
3`-region in pVL1393 and a synthetic oligonucleotide
(5`-CCACTTGTTCCCCTCTGCCTCCTGTCC-3`) that also spans the desired
mutation site in the mMCP-7 cDNA but in the opposite direction. The two
PCR-generated products were purified and mixed in equal proportions,
and then a third PCR was performed with the pVL1393-specific
oligonucleotides. The final PCR product was digested with EcoRI and inserted into EcoRI-digested pVL1393, and
the DNA sequence of the mutated construct was confirmed in both
directions. Synthetic oligonucleotides
5`-CAGTACCTCTATTACGAGGACCACCTGATGACT-3` and
5`-AGTCATCAGGTGGTCCTCGTAATAGAGGTACTG-3`,
5`-CTCTATTACCATGACGAGCTGATGACTGTGAGC-3` and
5`-GCTCACAGTCATCAGCTCGTCATGGTAATAGAG-3`,
5`-AGCCAGATCATCACAGAGCCCGACTTCTACATC-3` and
5`-GATGTAGAAGTCGGGTCTTGTGATGATCTGGCT-3`, and
5`-GCTGGGAATGAAGGAGAAGACTCCTGCCAG-3` and
5`-CTGGCAGGAGTCTTCTCCTTCATTCCCAGC-3` were used to generate the
His
Glu
, His
Glu
, His
Glu
, and
His
Glu
mutants of pro-mMCP-7,
respectively.
To
determine whether any of the recombinant pro-mMCP-7 produced by
infected High Five cells was N- or O-glycosylated,
SDS-PAGE buffer was added to each medium and cell pellet. Samples were
briefly sonicated and then boiled for 5 min. A 50-µl portion of
7.5% Nonidet P-40 was added to each 100-µl sample, followed by the
addition of 2 µl (0.5 milliunits) of Flavobacterium
meningosepticumN-glycanase (Genzyme) with or without 2
µl (
2 milliunits) of Diplococcus pneumoniaeO-glycanase (Genzyme). After the samples were incubated
overnight at 37 °C, the resulting digests were subjected to
SDS-PAGE/immunoblot analysis to assess for an N- or O-glycanase-induced change in the size of recombinant
pro-mMCP-7.
Figure 1: Amino acid alignment of mMCP-7 and bovine pancreatic trypsin. The numbers in the top line refer to the residues in mMCP-7. The five horizontal lines under the alignment indicate the sequences that comprise the positively charged, heparin-binding region in mMCP-7. The Arg, Lys, and His residues in this region are highlighted. The stars indicate the five His residues that were individually mutated to Glu.
Figure 2:
Three-dimensional model of mMCP-7 at pH
5.5. In the ribbon diagram (A), the positively charged side
chains (Lys, Arg, and His), the C atoms of the
negatively charged residues (Asp, Glu), the active site side chains
(His
, Asp
, and Ser
), and the
mutated His side chains are shown in blue, red, yellow, and green,
respectively. The figure was prepared by programs MOLSCRIPT (40) and RASTER3D(41) . The His, Arg, Lys, and active
site residues are numbered in pink. The space-filling model of the
heparin-binding region (B) has been obtained by rotating 90
degrees the model depicted in panel A. His residues 8, 27, 68,
70, 80, 108, and 187 are shown in green. The rest of the residues are
shown in white.
Figure 3:
Electrostatic potential at the positively
charged region of normal and altered mMCP-7. Non-mutated mMCP-7 (A) and the HisGlu
mutant of
mMCP-7 (B) at the acidic pH of the granule. The molecular
surfaces of the models are colored by the electrostatic potential, as
shown by the color bar on each panel (in units of kT; 1 kT unit
= 0.58 kcal/electron mol). The figures were prepared by program
GRASP(42) , using the relative dielectric constants of 2 and 78
for protein and solvent, respectively, and the salt concentration of
150 mM. The three His residues in the positively charged
region are numbered in black. The orientation of the tryptase is the
same as in Fig. 2B.
To
determine whether the positively charged region in mMCP-7 is likely to
bind to the heparin chain of serglycin proteoglycan, it is necessary to
calculate the overall electrostatic potential, which is a sum of the
contributions from positive and negative charges from all parts of the
molecule. A plot of the electrostatic potential around mMCP-7 with
positively charged His residues is shown in Fig. 3A.
There is only one region of pronounced positive electrostatic potential
and the electrostatic potential at the molecular surface of this region
is diminished significantly at neutral pH when the positive charges on
the His residues are eliminated (data not shown). Moreover, the
positive electrostatic potential of this region is also greatly
diminished if His, His
(Fig. 3B), or His
is mutated to Glu.
In contrast, His
and His
do not reside in
the positively charged region, and their conversion to Glu does not
influence the electrostatic potential of the region (data not shown).
Figure 4: Expression of pro-mMCP-7 in High Five cells. High Five cells were either non-infected or were infected with the pVL1393 construct that encodes prepro-mMCP-7. Three days later, samples of the cell pellets (C) and culture supernatants (S) were applied to individual wells of two polyacrylamide gels. After SDS-PAGE, the gel on the left was stained with Coomassie Blue. On the right, the protein blot from the duplicate gel was stained with anti-mMCP-7 Ig. Molecular mass markers in kDa are indicated on the left.
Figure 5:
Effect of pH on the binding of recombinant
pro-mMCP-7 to heparin-Sepharose CL-6B. In this experiment, the culture
supernatant from infected High Five cells was adjusted to pH 5.0 and
applied to the heparin affinity column. The column was sequentially
washed with phosphate-buffered saline adjusted to pH 5.0, 4.5, 5.0,
5.5, 6.0 (lanes 3-5), 6.5 (lanes 6-8),
and 7.0 (lanes 9-15). The gel depicted in panel A was stained with Coomassie Blue. The SDS-PAGE/immunoblot depicted
in panel B was prepared from a second gel and was stained with
anti-mMCP-7 Ig. No 28-kDa immunoreactive protein eluted when the
affinity column was washed with pH 4.5, 5.0, or 5.5 buffer (data not
shown). Lane 2 contains a sample of the culture supernatant
that was initially applied to the column. Molecular mass markers in kDa
are indicated on the left and in lane 1 of panel
A.
Figure 6:
Effect of NaCl concentration on the
binding of recombinant pro-mMCP-7 to heparin-Sepharose CL-6B. Cultured
supernatant from infected High Five cells was adjusted to pH 5.2 and
applied to the heparin affinity column. The column was washed with pH
5.2 buffer before the start of the salt gradient. The proteins
recovered in the column fractions were separated by SDS-PAGE and
transferred to Immobilon, and the resulting protein blot depicted in panel A was stained with anti-mMCP-7 Ig. Lanes 1, 2,
and 3 consist of molecular weight standards (Std.), a
sample of the culture supernatant (CS) before application to
the column, and the unbound proteins in the fall-through fraction (FT), respectively. Fractions 4-15 represent the initial
gradient fractions. pro-mMCP-7 begins to elute at a NaCl concentration
of 240 mM. The duplicate SDS-PAGE gel depicted in panel B was stained with Coomassie Blue. Molecular mass
markers in kDa are indicated on the left and in lane 1 of panel B.
Figure 7:
Binding of normal pro-mMCP-7 and five
HisGlu mutants of pro-mMCP-7 to heparin-Sepharose-6B. Samples of
the cultured supernatants from High Five cells induced to express
normal non-mutated pro-mMCP-7 (lanes 1 and 2), or the
His
Glu
(lanes 3 and 4),
His
Glu
(lanes 5 and 6), His
Glu
(lanes 7 and 8), His
Glu
(lanes
9 and 10), or His
Glu
(lanes 11 and 12) mutants of pro-mMCP-7 were
adjusted to pH 5.0 and applied to replicate columns of
heparin-Sepharose CL-6B. The starting supernatants (Sup.) (lanes 1, 3, 5, 7, 9, and 11) and the non-bound
proteins in the fall-through fractions (FT) (lanes 2, 4,
6, 8, 10, and 12) were analyzed for the presence of
immunoreactive mMCP-7. Molecular mass markers in kDa are shown on the left.
Figure 8:
Sepharose CL-2B chromatography at neutral
pH of immunoreactive mMCP-5 and mMCP-7 released from
FcRI-activated mBMMC. Supernatants from Fc
RI-activated mBMMC
were chromatographed on a Sepharose CL-2B column, and samples of
fractions 5-39 were assessed for their presence of immunoreactive
mMCP-5 (A) and mMCP-7 (B). Fractions 13 and 14 and
fractions 34-39 represent the column's excluded and total
volumes, respectively. A sample of starting supernatant (St.)
before gel filtration chromatography was also assessed for the presence
of mMCP-5 and mMCP-7. Similar findings were obtained in three other
experiments.
At the granule pH of 5.5(43) , mMC-CPA and all mMCPs are positively charged proteins. Depending on their cytokine microenvironment, mouse mast cells will synthesize either chondroitin sulfate E or heparin onto the serglycin proteoglycan peptide core(44) . Because chondroitin sulfate E and heparin are two of the most negatively charged molecules in the mouse, it has been assumed that mMC-CPA and most, if not all, mMCPs are ionically bound to serglycin proteoglycans within the granule. To investigate the interaction of mMCP-7 with heparin-containing serglycin proteoglycans inside and outside the mast cell, this tryptase was modeled, expressed in insect cells, and studied by site-directed mutagenesis.
Cathepsin
G is a chymase serine protease that, like mMCP-7 in the mouse (45) , is found in the secretory granules of human skin mast
cells(46) . Based on biosynthetic radiolabeling techniques, it
takes 90 min for newly translated cathepsin G to be converted to
active protease in U937 cells (47) and in transfected rat
basophil leukemia mast cells(48) . If it is assumed that its
signal peptide is enzymatically removed in the endoplasmic reticulum,
then the prolonged time it takes to generate mature cathepsin G
suggests that the final post-translational maturation of this and other
granule proteases in mast cells occurs after the zymogens are targeted
to granules. In agreement with this conclusion, Dikov and co-workers (32) found small but significant amounts of pro-mMC-CPA in the
secretory granules of cultured mouse mast cells. Certain mast cell,
myeloid, and lymphoid granule serine proteases are activated by the
granule-localized thiol protease, dipeptidyl peptidase
I(49, 50) . Moreover, recombinant human mast cell
pro-chymase can be efficiently activated by dipeptidyl peptidase I only
after the zymogen binds to heparin(33) . Heparin biosynthesis
is completed relatively late during the post-translational modification
of serglycin proteoglycan peptide core in the trans-region of
the Golgi(51) . These and other studies suggest that the
chymase and mMC-CPA zymogens bind to serglycin proteoglycans during or
shortly after exit from the Golgi and are slowly converted to active,
mature enzymes in the granule.
Because mMC-CPA and the mast
cell chymases are first packaged in granules as zymogens, it was
hypothesized that the mast cell tryptase, mMCP-7, also is first
packaged in granules as a zymogen. Thus, mMCP-7 was expressed in its
pro form rather than its ``mature'' form. In preliminary
experiments, both COS-1 cells and rat basophil leukemia mast cells were
transfected with a pSR expression construct containing a
full-length mMCP-7 cDNA. However, because the amount of recombinant
protease obtained in these transfected mammalian cells was insufficient
for conducting extensive biochemical studies, we turned to insect cell
expression systems. As shown in Fig. 4, High Five cells produced
and secreted large amounts of soluble 27- to 32-kDa proteins that were
recognized by anti-mMCP-7 Ig. NH
-terminal amino acid
analysis indicated that all immunoreactive proteins secreted into the
culture medium and all immunoreactive cell-associated proteins were
pro-mMCP-7. In BALB/c mBMMC, mMCP-7 exhibits considerable size
heterogeneity due to its variable Asn-linked carbohydrate
content(36) . N- and O-glycanase analyses
revealed that recombinant pro-mMCP-7 was heterogeneous in its size
primarily because of variable glycosylation. Insect cell-derived
pro-mMCP-7 bound to the heparin-affinity column at pH <6.5 (Fig. 5) and NaCl concentration <0.24 M (Fig. 6). Since the pH of the granule in the mast cell has
been estimated to be
5.5(43) , these data support the
hypothesis that pro-mMCP-7 binds to serglycin proteoglycans inside the
granule.
All mouse mast cell proteases eventually are stored for long periods of time in the secretory granule in their mature, enzymatically active states(1, 2, 3, 4, 5) . How the mast cell minimizes autolysis of its granule proteases and prevents degradation of other granular constituents has not been determined. Although these proteases are optimally active at neutral pH, many of them possess significant enzymatic activity at pH 5.5(18, 52) . Skin mast cells were first discovered because of the affinity of their heparin-rich (53) secretory granules for cationic dyes(54) . All hematopoietic cells that store biologically active proteins in their secretory granules then were found to contain serglycin proteoglycans in this intracellular compartment. It has been presumed that, by binding to serglycin proteoglycans, the proteases are sterically prevented from degrading each other or any other large-sized protein that may be in the granule. Mast cell-derived heparin binds to numerous proteins in vitro that have a consensus amino acid sequence of X-B-B-X-B-X, or X-B-B-B-X-X-B-X, where X and B are non-charged and positively charged amino acids, respectively(55) . Another heparin-binding motif is the sequence, Trp-Ser-X-Trp(56) . However, none of these amino acid sequences are present in mMCP-7 (Fig. 1) or in any other known mouse mast cell granule protease. It has been suggested that His residues could play an important role in heparin binding to mast cell tryptases(13) . There are 14 His residues in mMCP-7, but these His residues appear to be randomly distributed throughout the amino acid sequence of the translated protein(11, 13) . A comparison of the mMCP-7 amino acid sequence with that of pancreatic trypsin (Fig. 1) does not indicate which His residues in mMCP-7 control its ionic binding to heparin.
A previous protein modeling study predicted that the
chymases mMCP-4 and mMCP-5 have two regions with net positive charges
ranging from +6 to +10(23) . The regions are located
at the opposite ends of the molecule and are away from the
substrate-binding cleft. It also was predicted that the chymases mMCP-1
and mMCP-2 have one of these regions. We now show that mMCP-7 has a
positively charged region at approximately the same location as the
four chymases ( Fig. 2and 3A). The mast cell chymases
have 0 to 1 His residue in their positively charged regions, whereas
mMCP-7 has 5 His residues. Thus, the potential in the positively
charged region in mMCP-7 is diminished substantially when the positive
charge on its His residues is eliminated at neutral pH. Moreover, the
positive electrostatic potential of this region is greatly diminished
if His, His
(Fig. 3B), or
His
is mutated to Glu. Because these three His residues
are predicted to be important for heparin binding, they were studied by
site-directed mutagenesis. In contrast, His
and
His
do not reside in the positively charged region, and
their conversion to Glu does not influence the electrostatic potential
of the region. Because these two His residues were predicted not to be
important for heparin binding, they were used as controls in the
site-directed mutagenesis study. Site-directed mutagenesis confirmed
that a mutation of any of the three His residues in the identified
positively charged region into Glu abolishes the binding of recombinant
pro-mMCP-7 to the heparin-affinity column at pH 5.0-5.5 (Fig. 7). The substitution of the two His residues outside the
positively charged region does not affect this binding. It is possible
that the mutations caused a global change in the three-dimensional
structure of pro-mMCP-7. However, generally the mutation of a single
charged amino acid that resides on the surface of a protein will not
cause a global change in its three-dimensional
structure(57, 58) . Moreover, because similar findings
were obtained if His
, His
, or His
were mutated (Fig. 7), it is highly unlikely that each of
these mutations would cause the same global change in the
three-dimensional structure of the protein. Thus, the functional
differences among the mutants are most likely due to the charge
differences between the mutated side chains.
The modeling study and experiments with recombinant pro-mMCP-7 indicate that the tertiary structure of mMCP-7 is critical for enabling the mast cell to store large amounts of the enzymatically active protease in its granules. A binding site consisting of several segments that are brought together in the folded protein is highly advantageous because it prevents the mast cell from storing denatured protease in its granules. Inasmuch as mast cells, monocytes/macrophages, natural killer cells, cytotoxic lymphocytes, basophils, eosinophils, neutrophils, and platelets/megakaryocytes all contain positively charged proteins in their granules along with negatively charged serglycin proteoglycans, the manner by which mMCP-7 is packaged in the granules of mast cells may represent a general control mechanism used by hematopoietic effector cells to package biologically active proteins in high concentration in a small number of secretory granules.
The mast
cells in the ear and skin of the BALB/c mouse and the
interleukin-3-dependent mBMMC developed in vitro from this
strain all express mMCP-5, mMCP-6, mMCP-7, and mMC-CPA. When mBMMC are
activated through their FcRI receptors, they exocytose nearly all
of their granule mMC-CPA and
80% of their granule serine proteases
as a >10
Da macromolecular complex with
heparin-containing serglycin proteoglycans(1, 15) .
Three-dimensional modeling of four mouse mast cell chymases predicted
that their heparin-binding domains are comprised predominantly of Lys
and Arg residues rather than His residues. These findings explain why
the mast cell chymases fail to dissociate from serglycin proteoglycan
outside the mast cell at neutral pH. The three-dimensional modeling and
heparin-interaction studies with recombinant pro-mMCP-7 predicted that
mMCP-7 would differ from other proteases in that it would dissociate
from the macromolecular complex exocytosed from mast cells. Thus, the
status of mMCP-7 released from immunologically activated mBMMC was
re-examined. We now report that native mMCP-7, but not mMCP-5,
dissociates at neutral pH from the endogenous macromolecular complex
exocytosed from activated mBMMC (Fig. 8).
Based on its amino
acid sequence (10) and three-dimensional model(23) ,
mMCP-5 is likely to be a chymase with specificity for aromatic and/or
aliphatic amino acids. In contrast, mMCP-7 is likely to be a tryptase
with specificity for basic residues at the P1
position(11, 13) . Since mMC-CPA (1) and rat
MC-CPA (18) are exopeptidases that prefer carboxyl-terminal
aromatic and aliphatic amino acids, the continued physical association
of mMCP-5 (Fig. 8A) and mMC-CPA (1) in the
macromolecular complex outside the mast cell may be a mechanism by
which these two neutral proteases coordinate degradation of common
protein substrates. In addition, because of its large physical size,
the macromolecular complex may represent a way to retain enzymatically
active mMCP-5 and mMC-CPA around the mast cell in inflamed tissue
sites. mMCP-7 is likely to be quite different from mMCP-5 in its
substrate specificity. Thus, there would be no need for mMCP-7 to
remain physically associated with mMC-CPA outside the mast cell if
these proteases degrade distinct proteins. Because mMCP-7 dissociates
from the protease-proteoglycan complex outside FcRI-activated
mBMMC (Fig. 8B), it is possible that this tryptase is
not retained for long periods of time in the inflamed sites in the ear
and skin where mast cells containing mMCP-7 reside. It was recently
discovered that the liver and spleen mast cells in the V3 mastocytosis
mouse express every known mast cell protease, but only mMCP-7 is found
in high concentrations in the peripheral blood 20 min after the V3
mastocytosis mouse is systemically activated with IgE and antigen. (
)Most studies carried out to deduce the functions of the
mast cell's tryptases have focused on their potential biologic
effects in the tissue site immediately adjacent to the activated mast
cell. If mMCP-7 is able to retain its enzymatic activity for a
substantial length of time after its dissociation from the
macromolecular complex, this tryptase could degrade proteins at sites
distant from the activated mast cell. Thus, the possible systemic
effects of mMCP-7 must be considered in future functional studies of
this tryptase.