From the Department of Medicine, Brigham and
Women's Hospital, and Department of Medicine, Harvard Medical School,
Boston, Massachusetts 02115, ¶ Department of Medicine,
University of New South Wales, and Department of Immunology, Allergy,
and Infectious Disease, St. George Hospital, Kogarah,
New South Wales 2217, Australia,
Rockefeller
University, New York, New York 10021, and ** Department of
Pediatrics, Division of Pulmonary Medicine, Allergy, and Clinical
Immunology, Children's Hospital Medical Center, University of
Cincinnati Medical Center, Cincinnati, Ohio 45229
Received for publication, November 16, 2000, and in revised form, March 1, 2001
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Genomic blot analysis raised the
possibility that uncharacterized tryptase genes reside on chromosome 17 at the complex containing the three genes that encode mouse mast cell
protease (mMCP) 6, mMCP-7, and transmembrane tryptase (mTMT). Probing
of GenBank's expressed sequence tag data base with these three
tryptase cDNAs resulted in the identification of an expressed
sequence tag that encodes a portion of a novel mouse serine protease
(now designated mouse tryptase 4 (mT4) because it is the fourth member
of this family). 5'- and 3'-rapid amplification of cDNA ends
approaches were carried out to deduce the nucleotide sequence of the
full-length mT4 transcript. This information was then used to clone its
~5.0-kilobase pair gene. Chromosome mapping analysis of its gene,
sequence analysis of its transcript, and comparative protein structure
modeling of its translated product revealed that mT4 is a new member of the chromosome 17 family of mouse tryptases. mT4 is 40-44% identical to mMCP-6, mMCP-7, and mTMT, and this new serine protease has all of
the structural features of a functional tryptase. Moreover, mT4 is
enzymatically active when expressed in insect cells. Due to its 17-mer
hydrophobic domain at its C terminus, mT4 is a membrane-anchored tryptase more analogous to mTMT than the other members of its family.
As assessed by RNA blot, reverse transcriptase-polymerase chain
reaction, and/or in situ hybridization analysis, mT4 is expressed in interleukin-5-dependent mouse eosinophils, as
well as in ovaries and testes. The observation that recombinant mT4 is
preferentially retained in the endoplasmic reticulum of transiently transfected COS-7 cells suggests a convertase-like role for this integral membrane serine protease.
A complex of genes resides on human chromosome 16p13.3 that
encodes the homologous tryptases The amino acid sequences of mMCP-6 and mMCP-7 are ~75% identical,
and the mast cells (MCs) in the skin and skeletal muscle of BALB/c mice
express both tryptases (12, 13). Nevertheless, in vivo and
in vitro studies have established that these two homologous serine proteases are metabolized quite differently during allergic reactions (12). Moreover, they are functionally distinct. Because fibrinogen is a physiologic substrate of mMCP-7 (14), this tryptase appears to help prevent the deposition of fibrin/platelet clots in
inflammatory sites so that the various granulocytes and lymphocytes in
the blood are not physically prevented from entering an inflamed mouse
tissue site. Recent data have suggested that mMCP-7 also regulates
eosinophil extravasation in
tissues.3 MCs are essential
for combating bacterial infections in the peritoneal cavity (15-17).
Because peritoneal MCs express mMCP-6 (11, 18), because the complement
factors C3a and C5a induce peritoneal MCs to degranulate (19), and
because recombinant mMCP-6 is a potent and selective inducer of
neutrophil extravasation into the peritoneal cavity (20), mMCP-6
appears to play an important and protective role in bacterial
infections. The function of mTMT is not yet known, but its C-terminal
hydrophobic domain (7) probably causes its prolonged retention at the
plasma membrane when mTMT+ MCs are immunologically activated.
Because our previous genomic blot analyses suggested the presence of
undiscovered tryptase-like genes in the mouse genome and because mouse
chromosome 17A3.3 to 17B1 has not been sequenced in its entirety, we
used a gene-hunt approach in the present study to identify a new member
of this important family of serine proteases. We now describe the
isolation, characterization, and expression of the fourth member of the
mouse tryptase family of serine proteases.
Cloning of the mT4 cDNA and Gene--
The nucleotide
sequences of the mTMT, mMCP-6, and mMCP-7 transcripts were used as
templates to search for novel, but related, mouse tryptase-like ESTs in
the GenBankTM data base. Sequence analysis of the mouse
testis-derived clone AI326140, obtained from the "Integrated
Molecular Analysis of Gene Expression" consortium, revealed that its
insert corresponded to a portion of what appeared to be a novel serine
protease. Based on the deduced nucleotide sequence of this clone, 5'-
and 3'-rapid amplification of cDNA ends (RACE) approaches were
carried out on a pool of testis cDNAs
(CLONTECH) to deduce the nucleotide and amino acid
sequences of the full-length transcript. The 3'-RACE reaction was
carried out with 5'-CACCTACAATAACTTCATCCAGC-3' and the anchor
oligonucleotide 5'-CCATCCTAATACGACTCACTATAGGGC-3'. The resulting DNA
products were purified on a 1% agarose gel, subcloned into pCR2.1
(Invitrogen, Carlsbad, CA), and the inserts in two of the arbitrarily
selected clones were sequenced. The 5'-RACE was carried out with the
RLM-RACE kit (Ambion, Austen, TX). The mT4-specific primer
5'-GACTGGATCCAGTTGTAGTGATGACTG-3' and the outer primer
5'-GCTGATGGCGATGAATGAACACTG-3' were used in the first PCR. One
microliter of the generated product was then used as a template in the
second PCR step with the mT4-specific primer
5'-CCCAGCAGTCAGTTCGGTTCTC-3' and the inner primer
5'-CGCGGATCCGACACTCGTTTGCTGGCTTTGATGAAA-3'. The resulting products were
purified and subcloned, and the inserts in six of the arbitrarily
selected clones were sequenced.
Because all mouse and human tryptase genes so far cloned in this family
are <6 kb in size, a long range nested PCR approach was used to
isolate and characterize the mT4 gene from BALB/c mice. The
oligonucleotides 5'-GCAAGACGTTGGTGCCACTGCTG-3' and
5'-GTGACGTACACGTGTGGGCTCAGGCAG-3' were used in the initial 30-cycle
PCR, whereas the oligonucleotides 5'-ATGGCCTTACAGTCAACCTATTTGCAG-3' and
5'-GCCTGAGCAGCCCATTGCGGATC-3' were used in the subsequent
28-cycle reaction. Recombinant Thermus thermophilus DNA
polymerase (PerkinElmer Life Sciences) was used in both PCRs.
Genomic Blot Analysis and Chromosomal Location of the mT4
Gene--
Some of the human tryptases that have been cloned during the
last decade are ~98% identical (3). Thus, to determine whether or
not the mouse genome contains other genes that closely resemble the
mT4 gene, replicate 25-µg samples of BALB/c mouse genomic DNA were digested separately at 37 °C for ~15 h with
EcoRI, DraI, BamHI, AvrII,
EcoRV, BglII, or HindIII (New England
Biolabs, Beverly, MA). The digests were fractionated on a 1% agarose
gel, and the separated fragments were blotted onto a MagnaGraph nylon
membrane (Micron Separations Inc., Westborough, MA). The resulting DNA blot was incubated for 2 h at 65 °C in QuikHyb solution
(Stratagene, La Jolla, CA) containing a 32P-labeled 536-bp
probe corresponding to the 5' end of the mT4 cDNA. The blot was
washed twice at room temperature for 20 min each in 2× SSC containing
0.1% SDS and then twice at 65 °C for 20 min each in 0.2× SSC
containing 0.1% SDS before being exposed to BIOMAX film.
A fluorescent in situ hybridization (FISH) technique was
used by Human Genome Systems (St. Louis, MO) to determine the
chromosomal location of the mT4 gene. Preliminary studies
revealed that the mT4 gene resided in the company's BAC
mouse genomic clone F1062. Slides containing normal metaphase
chromosomes derived from mouse embryonic fibroblasts were therefore
incubated with the digoxigenin dUTP-labeled clone F1062 in the presence
of 50% formamide, 10% dextran sulfate, 2× SSC, and sheared mouse
DNA. After this hybridization step, the slides were incubated with
fluoresceinated anti-digoxigenin antibody and counterstained with
4,6-diamidino-2-phenylindole.
Expression of mT4 at the mRNA Level--
To determine which
tissues in the mouse contain abundant levels of mT4 mRNA, a blot
(CLONTECH) containing ~2 µg of
poly(A)+ RNA from mouse heart, brain, spleen, lung, liver,
skeletal muscle, kidney, and testis was probed under conditions of high
stringency with a radiolabeled 536-bp probe that corresponded to the 5'
end of the mT4 transcript. After the probe was random primed with [
BALB/c mice exhibit a substantial, but transient, T
cell-dependent increase in the number of eosinophils and
MCs in their jejunum 7 and 14 days, respectively, after the animals are
infected with Trichinella spiralis (22). To determine
if the eosinophils or MCs that develop in the jejunum also contain mT4
mRNA, total RNA was isolated from the small intestine, spleen, and
liver of day-7 helminth-infected mice (n = 2) and
day-14 helminth-infected mice (n = 2). RT-PCR was then
used to determine whether or not any of the tissue specimens contained
mT4 mRNA. In this assay, the RT step was carried out at 55 °C
for 30 min. Forty five cycles of PCR were performed with the primers
5'-CAACAGCATGTGTAACCATATG-3' and 5'-GCCTGAGCAGCCCATTGCGGATC-3'; each
cycle consisted of a 5-s denaturing step at 94 °C, a 5-s annealing
step at 60 °C, and a 15-s extension step at 72 °C. This RT-PCR
approach also was used to evaluate mT4 expression in the ovaries and
testis before and after sexual maturation. In these reactions, 32 cycles were carried out rather than 45.
Location of mT4 mRNA-expressing Cells in the Testis--
For
the non-radioactive in situ hybridization approach, the
mT4-specific oligonucleotide
5'-CTATTTGGTAACGGTTGGAATAGGCCTGTAGGTTCCAGAGAGATGGCCTGG-3' was
labeled with digoxigenin-alkaline phosphatase with a
commercially available 3' end labeling kit (Roche Molecular
Biochemicals). BALB/c mouse testis was fixed in 4% paraformaldehyde in
0.1 M phosphate-buffered saline (PBS) at 4 °C. The
preparation was washed twice with PBS containing 2% dimethyl sulfoxide
and then dehydrated and embedded in JB4 glycomethacrylate according to
the manufacturer's instructions (Polysciences Inc., Warrington, PA).
Sections were cut on a Reichert-Jung Supracut microtome (Leica,
Deerfield, IL) at 5-µm thickness and picked up on glass slides. The
slides were incubated sequentially for 15 min at 37 °C in 0.025%
trypsin containing 2 mM calcium chloride and then for 30 min in Target Retrieval solution (Dako, Carpinteria, CA) at 95 °C.
After the slides were washed three times in distilled water, they were
incubated in prehybridization solution containing 50% formamide, 4×
SSC, 1× Denhardt's solution, sonicated salmon sperm DNA, and 10%
dextran sulfate at 42 °C for 30 min. The prehybridization solution
was removed; 50 µl of hybridization buffer containing the
digoxigenin-labeled oligonucleotide was added, and each specimen was
incubated overnight at 42 °C. The next morning, each slide was
washed twice with 2× SSC, once with 1× SSC at room temperature, and
then incubated with anti-digoxigenin-biotin complex for 30 min at room
temperature. The slides were washed twice with Tris-buffered saline,
incubated with streptavidin complex conjugated to horseradish
peroxidase, and washed twice again with Tris-buffered saline. Color
development was performed according to the manufacturer's instructions
(Dako). In this analysis, cells that contained abundant levels of mT4 mRNA stain brown.
A radioactive in situ hybridization approach was used to
confirm the obtained data with the above digoxigenin-alkaline
phosphatase-labeled probe. For this second type of in situ
hybridization analysis, nucleotides 1-536 of the mT4 cDNA (Fig. 1)
were subcloned into pCR2.1 in both the sense and antisense directions.
The resulting two plasmid DNA samples were linearized with
SpeI (New England Biolabs) and transcribed with T7 RNA
polymerase (Promega, Madison, WI) in the presence of
[ Expression of mT4 Protein in COS-7 Cells and Insect Cells and
Protein Modeling of Its Translated Mature Product--
To address
whether or not the mT4 transcript encodes an enzymatically active,
membrane-anchored protease, the entire coding region of mT4 cDNA
was placed in the expression vector pcDNA3.1/V5-His-TOPO (Invitrogen). This expression vector was chosen because the resulting product will contain the 14-mer V5 peptide
(Gly-Lys-Pro-Ile-Pro-Asn-Pro-Leu-Leu-Gly-Leu-Asp-Ser-Thr) at its C
terminus, thereby allowing its detection with anti-V5 antibody. Vector
lacking an insert was used as a negative control in the transfection
experiments. African green monkey, SV40-transformed kidney COS-7 cells
(line CRL-1651, ATCC, Manassas, VA) were cultured in Dulbecco's
modified Eagle's medium containing 10% fetal bovine serum. Transient
transfections were performed with SuperFect (Qiagen, Valencia, CA)
according to the manufacturer's instructions. Cells were plated at a
density of 2 × 105 cells/well in 6-well plates
24 h prior to transfection. After the cells were transfected for
2-3 h, they were trypsinized, and portions of the resulting cells were
placed in 24-well plates containing 11-mm coverslips for
immunofluorescence microscopy. The remainder of the cells were placed
in 12-well plates for SDS-PAGE/immunoblot analysis. Conditioned media
and cells were collected 24 and 48 h post-transfection.
For immunofluorescence microscopy, transfected COS-7 cells grown on
coverslips were washed once with PBS, fixed in 4% paraformaldehyde for
10 min, and permeabilized in methanol for 10 min. The treated cells
were washed three times with PBS. They were then exposed to 10% donkey
serum in PBS for 1 h at room temperature to prevent nonspecific
binding of the relevant mouse and rabbit antibodies. After this step,
the cells were stained with mouse anti-V5 antibody (Invitrogen) in the
absence or presence of a mixture of rabbit anti-calnexin and
anti-calreticulin antibodies (StressGen, Victoria, British Columbia,
Canada). Calnexin and calreticulin reside in the endoplasmic reticulum
(ER). Thus, antibodies directed against these two proteins were used to
confirm the ER location of recombinant mT4 protein in the
transfectants. Cy®2-conjugated donkey anti-mouse antibody (Jackson
ImmunoResearch, West Grove, PA) was used to detect the mT4-V5 fusion
protein, whereas Cy®3-conjugated donkey anti-rabbit antibody (Jackson
ImmunoResearch) was used to detect calnexin and calreticulin. Stained
cells were viewed with a Nikon Eclipse 800 microscope. Images were
digitally captured using a CCD-SPOT RT camera and compiled using Adobe
Photoshop software.
For SDS-PAGE/immunoblot analysis, samples of the transfected COS-7
cells and their conditioned media were boiled in SDS sample buffer
containing
Other members of the tryptase family contain N-linked
glycans (23, 24). Analysis of the predicted primary amino acid sequence of mT4 revealed four potential N-linked glycosylation sites.
Thus, to determine if mT4 contains N-linked glycans, a
sample of the COS-7 cell-derived recombinant material was incubated
with PNGase F (New England Biolabs) using the manufacturer's suggested
reaction conditions. The resulting digest was then subjected to
SDS-PAGE/immunoblot analysis.
As noted under "Results," mT4 can be generated in COS-7 cells using
the above expression strategy. Unfortunately, because mT4 remains
tightly associated with the ER membrane of the cell, contaminating ER
proteins cannot be removed easily to evaluate the enzymatic activity of
the generated recombinant protein. We previously generated
pseudozymogen forms of mMCP-6 and mMCP-7 in High Five insect cells that
in each instance contain an enterokinase-susceptibility sequence
between the natural propeptide and catalytic domain (14, 20). Because
each pseudozymogen also contains the FLAG peptide Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys at its C terminus, it can be purified using an anti-FLAG antibody immunoaffinity column. Thus, to generate a
bioengineered form of recombinant mT4 that can be purified to near
homogeneity for functional studies, an expression construct was created
in which the C-terminal membrane-spanning domain of mT4
(i.e. residues 249-270) was replaced by the 8-residue FLAG peptide. Insect cells might be unable to activate constitutively the
generated product. To guard against that possibility, an
enterokinase-susceptibility sequence also was created in the construct
by inserting the 5-residue peptide Asp-Asp-Asp-Asp-Lys in between the
natural propeptide of mT4 and the first Ile residue in its catalytic
domain. In this way, the purified pseudozymogen could be activated by
enterokinase if needed. High Five insects cells, cultured in serum-free
media, were induced to express the bioengineered form of mT4 as
described previously (14, 20, 25, 26) for other recombinant mouse and
human tryptases. Insect cell lysis buffer from PharMingen was used to
liberate the cell-associated recombinant mT4 from pelleted cells. After
a 30-min incubation on ice, the resulting lysates were centrifuged at
~20,000 × g for 30 min at 4 °C. The supernatant
was removed and passed through a 0.45-µm cellulose acetate filter.
The cleared supernatant was then loaded at 4 °C onto an ~1-ml
column containing anti-FLAG M1 antibody (Sigma). After the column was
washed with 250 ml of Tris-buffered saline, pH 7.0, 0.1 M
glycine, pH 3.5, buffer was used to elute bound mT4. Ten 1-ml fractions
were collected into tubes containing 20 µl of 1 M
Tris-HCl, pH 8.0. Samples of resulting fractions were analyzed by
SDS-PAGE for the presence of Coomassie Blue+ proteins and
for immunoreactive mT4.
Casein is a protease-sensitive protein. Thus, resorufin-labeled casein
(Roche Molecular Biochemicals) was used to determine whether or not
insect cell-derived mT4 is enzymatically active. In this assay, a
50-µl sample of the eluate from the above immunoaffinity column was
added to 50 µl of 5 mM calcium chloride and 10 mM Tris-HCl, pH 5.0, containing or lacking 0.4 unit of
enterokinase (New England Biolabs); the sample was then incubated at
37 °C for 2 h. To evaluate the enzymatic activity of the
resulting recombinant material, 100 µl of 0.1 M Tris-HCl,
pH 7.8, containing 10 mM calcium chloride and 200 µg of
resorufin-labeled casein was added. After an 18-h incubation at
37 °C, each reaction was stopped by the addition of 480 µl of 5%
trichloroacetic acid, followed by a 10-min centrifugation at
>8,000 × g. A 400-µl sample of the resulting
supernatant was mixed with 600 µl of 0.5 M Tris-HCl, pH
8.8, and the absorbance was measured at 574 nm. Because low molecular
weight resorufin-labeled peptides released from its substrate are not
precipitated by the trichloroacetic acid step, the concentration of
resorufin-labeled peptides in the supernatant is directly proportional
to the general proteolytic activity present in the sample. Trypsin
(Sigma) was used as a positive control in this enzymatic assay.
A three-dimensional model of the extracellular domain of mature mT4
(i.e. residues 1-256) was built by MODELLER (27, 28) as described for
other MC proteases (7, 12, 29, 30). The primary template for
comparative protein structure modeling was the crystal structure of
human tryptase Cloning and Sequence Analysis of mT4 cDNA--
When the mTMT,
mMCP-6, and mMCP-7 cDNAs were used as templates to screen
GenBank's EST data base, an EST (accession number AI326140) was
identified that was somewhat homologous to all three target tryptases.
The relevant EST was obtained from the Integrated Molecular Analysis of
Gene Expression consortium, and nucleotide sequence analysis of the
entire insert in the clone revealed that it encoded a portion of a
serine protease (designated mT4 because it is the fourth member of this
family of serine proteases) not previously described. 5'- and 3'-RACE
approaches (Fig. 1A) were
therefore carried out to deduce the nucleotide sequence of the missing
portion of the transcript (Fig. 1B). The full-length mT4
cDNA consists of ~1095 nucleotides, and the 5'- and
3'-untranslated regions (UTRs) of the transcript consist of 23 and >97
nucleotides, respectively. The putative translation-initiation codon
conforms to the sequence present in most eukaryotic transcripts
(32).
Analysis of mT4 at the Protein Level--
The deduced amino acid
sequence of its cDNA suggested that mT4 could be initially
translated as an ~36-kDa zymogen consisting of 324 amino acids (Fig.
1B). Hydropathy plot analysis (Fig. 1C) disclosed
that mT4 possesses an unusual hydrophobic domain in its C terminus at
residues 251-267. Phylogenetic analysis (Fig. 2A) of all known mouse
proteins revealed that mT4 is most closely related to mTMT. However,
the degree of identity is only 44%. Every tryptase in this family has
an N-terminal sequence of Ile-Val-Gly-Gly when its propeptide is
removed (Fig. 2B). Because the zymogen form of mT4 also
possesses this sequence near its initially translated N terminus, it is
predicted that the 54-residue prepropeptide is cleaved at the Arg-Ile
site indicated in Fig. 1B. In its mature form, the protein
portion of the catalytic domain of mT4 is predicted to have a molecular
mass of ~30 kDa. However, because the mature protein contains
four potential N-linked glycosylation sites at Asn116, Asn123, Asn156, and
Asn229, post-translationally modified mT4 is expected to be
somewhat larger in size in vivo.
When transfected into COS-7 cells, the level of immunoreactive mT4 in
the conditioned media of the transfectants was below detection by
SDS-PAGE/immunoblot analysis (Fig.
3A). Thus, very little, if
any, recombinant mT4 was constitutively released from the
transfectants. The fact that mT4 was preferentially recovered in the
microsomal fraction of the cell lysates (Fig. 3B) confirmed that mT4 is a membrane-anchored protein. Immunohistochemical analysis of the transfectants revealed that mT4 was preferentially retained in
the calexin/calreticulum-enriched ER (Fig. 3, D-F). The
fact that immunoreactive mT4 was not released after trypsin treatment (data not shown) confirmed that very little, if any, mT4 targets to the
plasma membrane in transfected COS-7 cells. As assessed by SDS-PAGE
analysis, an immunoreactive protein of ~40 kDa was identified in
COS-7 cell transfectants that shifted to ~35 kDa after PNGase F
treatment (Fig. 3C). Based on the magnitude of this change
in its molecular weight, mT4 contains more than one N-linked
glycan.
By using resorufin-labeled casein as a substrate, the level of
proteolytic activity in the lysates of the mT4-expressing COS-7 cells
was significantly greater than that in the lysates of control non-transfectants (data not shown). However, the amounts of active enzyme were low, and recombinant mT4 remained tightly associated with
the membrane of the ER (Fig. 3). Thus, a bioengineered form of mT4
possessing the FLAG peptide at its C terminus was expressed in High
Five insect cells (Fig. 4). In contrast
to what occurs when similar FLAG derivatives of mMCP-6 (20) and mMCP-7
(14) are expressed in insect cells, the FLAG derivative of mT4 was not
constitutively secreted from the insect cells. Nevertheless, the
recombinant protein could be purified from the lysates of its
expressing cells using the immunoaffinity column (Fig. 4A). Even though the amount of protein in fractions 5 and 6 of the eluate of
the column was below detection by Coomassie Blue staining of a
duplicate gel (data not shown), the recombinant mT4 in these fractions
exhibited substantial proteolytic activity in vitro (Fig.
4B). The fact that the enzymatic activity of the recombinant material did not increase dramatically following enterokinase treatment
indicated that much of the purified mT4 was constitutively active. At
present, we cannot ascertain whether activation of the mT4
pseudozymogen occurred inside the insect cells or during its
purification. Nevertheless, because more trypsin was used on a weight
basis in the depicted experiment, the broad enzymatic activity of
recombinant mT4 is at least as good as that of trypsin in regard to its
ability to degrade casein.
The overall fold of mature mT4 is predicted to be similar to that of
most serine proteases (Fig.
5A). For example, like all other functional serine proteases, mT4 possesses the conserved triad
(i.e. His41, Asp93, and
Ser194) in its putative catalytic site. Like mTMT, mT4
lacks a number of the Pro and Tyr residues (31) that are needed for
human tryptase Nucleotide Sequence and Chromosomal Location of the mT4
Gene--
Although two DNA fragments were detected when a blot
containing AvrII-, HindIII, or
BglII-digested mouse genomic DNA was probed with the mT4
cDNA (Fig. 6A), subsequent
nucleotide sequence analysis revealed that these findings were due to
the presence of internal enzyme restriction sites within the gene.
Thus, a single gene encodes mT4. The fact that no additional DNA
fragment was observed when the genomic blot was probed at moderate
stringency (data not shown) suggests that no closely related gene is
present in the mouse genome.
The initial FISH analysis revealed that the mT4 gene resides
on the proximal region of a small-sized chromosome believed to be mouse
chromosome 17. Based on that data, a second experiment was conducted in
which a probe specific for the telomeric region of chromosome 17 was
co-hybridized with the mT4-containing genomic clone. This experiment
resulted in the specific labeling of the telomere and the proximal
portion of mouse chromosome 17. Measurements of 10 specifically labeled
chromosomes 17 demonstrated that the mT4 gene is located at
a position that is 23% of the distance from the
heterochromatic-euchromatic boundary to the telomere of the chromosome.
This area corresponds to the interface between bands 17A3.3 and 17 B1
(Fig. 6B) where the mMCP-6, mMCP-7, and mTMT genes reside. When 80 metaphase cells were analyzed, 77 exhibited specific labeling.
With a long range PCR approach, the entire mT4 gene was
isolated and sequenced. The mT4 gene is ~5.0 kb in size
and contains 6 exons (Fig.
7A). All exon/intron splice
sites conformed to the GT/AG rule for other eukaryotic genes (33).
Exons 1-6 consist of 126, 27, 166, 284, 155, and 237 bp, respectively,
whereas introns 1-5 consist of 101, 291, 599, 2717, and 269 bp,
respectively. The exon/intron organization of the mT4 gene
differs somewhat from that of the other three mouse genes in this
family (Fig. 7B). For example, although the mT4 transcript
is similar in size to that of the other three mouse MC tryptase
transcripts, its gene is larger due primarily to intron 4. The
mT4 gene also has six exons, whereas the mTMT and
mMCP-7 genes have five exons.
Expression of mT4 in Immune and Non-immune Cells--
As assessed
by RNA blot analysis, the level of mT4 mRNA was below detection in
normal mouse bone marrow (Fig.
8A). Transgenic mice that have
been induced to express abnormally high levels of IL-5 exhibit a
constitutive eosinophilia (21). Although barely detectable amounts of
mT4 mRNA were found in the bone marrow of IL-5 transgenic mice,
larger amounts of this transcript were present in the
IL-5-dependent eosinophils purified from the transgenic animals (Fig. 8A). In confirmation of these data, mT4
mRNA also could be detected in the jejunum of a T. spiralis-infected BALB/c mouse (Fig. 8B) precisely when
the number of eosinophils are maximal in this tissue (34).
mT4 mRNA could not be detected by RNA blot analysis in normal
heart, brain, spleen, lung, liver, skeletal muscle, and kidney (Fig.
8C), as well as ear, tongue, stomach, and intestine (data not shown). Because these tissues contain substantial numbers of MCs,
mT4 is the only member of its family that is not preferentially expressed in MCs. The level of mT4 mRNA also was below detection in
day-7-17 mouse embryos (Fig. 8C). However, using an RT-PCR approach, mT4 mRNA was detected in the testes and ovaries of adult mice (Fig. 8D). As assessed by two different in
situ hybridization methods, mT4 is transiently expressed
relatively late in spermatogenesis during the cap phase of acrosome
formation (Fig. 9).
While at least six distinct tryptase genes reside at a complex on
human chromosome 16 (1-3, 6, 7), only three corresponding genes have
been identified so far on the syntenic region of mouse chromosome 17 (7-11). We now describe a new mouse gene in this family that is
related to the genes that encode mTMT, mMCP-6, and mMCP-7.
The mT4 cDNA (Fig. 1) and gene (Fig. 7) encode a 324-residue
polypeptide having a 54-residue prepropeptide and a 17-residue, C-terminal hydrophobic domain. When transiently expressed in COS-7 cells, mT4 remains cell-associated (Fig. 3). Thus, as predicted based
on analysis of its cDNA and gene, translated mT4 is a
membrane-anchored serine protease. Like the transmembrane protease
angiotensin-converting enzyme (35), the C-terminal hydrophobic domain
of mT4 is flanked by Asp and Arg residues. Because
angiotensin-converting enzyme (35), prostasin (36), and acrosin (37)
can be released from cells by a proteolytic processing mechanism, the
possibility has not been ruled out that under certain situations mT4
undergoes a similar post-translational processing event in
vivo to cause its release from cells.
The zymogen form of mT4 contains 10 Cys residues. Based on the crystal
structure of human tryptase Although its physiologic substrate(s) was not deduced in this initial
study, mT4 is enzymatically active when expressed in insect cells (Fig.
4). mT4 has several features that indicate that it probably exhibits
tryptic-like activity in vivo. For example, all tryptic
serine proteases possess an Asp six residues N-terminal of the
catalytic Ser residue. This Asp is needed for interaction of the serine
protease with the P1 Lys or Arg residue in the susceptible substrate
(41). Not only does mT4 have the conserved Asp at residue 188 (Figs. 1,
2A, 5, and 6), but also this residue is predicted to reside
at the base of the substrate-binding cleft. As in other tryptic
proteases, Gly215 and Gly225 are present in
mT4. Based on the crystal structures of rat trypsin (42) and human
tryptase When properly folded, a hydrophobic domain consisting of eight Trp
residues forms on the surface of every MC tryptase opposite that of the
substrate-binding cleft (31, 43). Expression/site-directed mutagenesis studies revealed that this domain is of functional importance in the maturation of mMCP-7 (26). The observation that mT4
contains these conserved residues (Trp12,
Trp14, Trp35, Trp128,
Trp132, Trp206, Trp214, and
Trp236) (Fig. 2B) and that they reside on the
appropriate surface region (Fig. 5A) is further evidence
that mT4 is a functional tryptase in vivo.
Genomic blot analysis (Fig. 6A) revealed that there is only
one mT4-like gene in the mouse genome, and FISH
analysis (Fig. 6B) revealed that this new gene resides on
mouse chromosome 17 quite close to the mMCP-6, mMCP-7, and
mTMT genes. Two transcripts that differ slightly in their
size were seen in the testis (Fig. 8C). Although the
possibility has not been ruled out that the mT4 transcript can undergo
alternative spicing in the testis, this seems unlikely because analysis
of its gene (Fig. 7A) predicts that a functional enzyme
would not be generated if any one of the 6 exons is deleted. Because
sequence analysis of the eight RACE products failed to reveal a
differentially spliced transcript, a more likely explanation for the
RNA blot data is that different transcription-initiation sites are used
to generate transcripts with variable sized 5'-untranslated regions.
The deduced amino acid sequence of mT4 is <45% identical to that of
mMCP-6, mMCP-7, and mTMT (Fig. 2). Although mT4 is most homologous to
human Esp-1 (hEsp-1), the sequence identity is still only 68%, and mT4
and hEsp-1 differ in a number of ways. At the protein level, hEsp-1
lacks the 3-residue cytoplasmic tail found in mT4 (Fig. 1B).
Cytoplasmic tails often regulate intracellular routing of membrane
proteins. hEsp-1 is a plasma membrane-anchored protease (4, 44). The
fact that recombinant mT4 is unable to reach the plasma membrane in
transfected COS-7 cells implies a regulatory role for the three
cytoplasmic residues in the ER retention of mT4. The membrane-spanning
domains of mT4 and hEsp-1 also differ substantially in their length and
primary amino acid sequences, as do their prepropeptides. More
important, the amino acid sequences that consist of 6 of the 7 loops
that form the substrate-binding clefts of mT4 and hEsp-1 are very
different (Figs. 2B and 5B). Thus, the preferred
substrate specificities of these two tryptases are most certainly
distinct in vivo. Although the mT4 (Figs. 8C and
9) and hEsp-1 (44) transcripts are present in abundance in the testis,
their precise location in this tissue also differs. hEsp-1 has been
reported to be expressed exclusively by primary spermatocytes before
their first meiotic division (44). In contrast, as assessed by two
different in situ hybridization methods, mT4 is transiently
expressed in secondary spermatocytes (Fig. 9). Although RNA blot (Fig.
8A) and RT-PCR (Fig. 8B) data indicate that
IL-5-dependent mouse eosinophils express mT4, the level of
the mT4 transcript in those mouse eosinophils also appears to be
considerably less than the level of hEsp-1 mRNA in human eosinophils (4) and human K562 leukemia
cells.4 Several tissues
(e.g. lung, pancreas, spleen, and bone marrow) that lack mT4
mRNA in mice (Fig. 8, B and C) contain hEsp-1
mRNA in humans (4, 44). Moreover, mT4 is expressed in both the testis and ovaries (Fig. 8D). Finally, at the genome level,
intron 4 is notably larger in the mT4 gene (2717 versus 1985 bp), whereas introns 2 and 3 are notably larger
in the hEsp-1 gene (291 versus 344 bp and 599 versus 710 bp, respectively) (Fig. 7B). The
regions of the mouse and human chromosomes where the tryptase complexes of genes reside have not yet been sequenced in their entirety. Nevertheless, based on all of the above differences, it is unlikely that mT4 is the mouse ortholog of hEsp-1/testisin. If it is the mouse
ortholog, substantial divergence occurred in this gene, its translated
product, its expression pattern, and presumably its substrate
preference during the last 40-100 million years of evolution.
Penetration of the egg zona pellucida by sperm is essential for
fertilization. Because various trypsin inhibitors can block sperm
penetration of the zona pellucida (45), one or more tryptic proteases
appears to play an essential role in fertilization. Acrosin is a major
tryptic protease in the testis. However, no fertilization defects were
observed in mice when the acrosin gene was disrupted (46). Thus, an
unidentified tryptase must play a more critical role in this biologic
process. Although mouse testicular serine protease (mTesp)-1 (47),
mTesp-2 (47), and mTesp-4 (48) were recently cloned from mouse testis
and found to reside in the acrosomal compartment of sperm, nothing is
known about their in vivo functions and substrate
specificities. The observation that each of these proteases possess an
Arg residue in its propeptide adjacent to the N-terminal Ile in the
mature enzyme suggests that an undefined tryptic-like enzyme is
required for the proteolytic processing of their propeptides.
Membrane-anchored, tryptic-like convertases such as human furin/PACE
and yeast Kex2 play important roles in the post-translational
processing of a diverse array of biologically active proteins (49).
Because mT4 is a membrane-anchored serine protease that resides in the ER of transfected COS-7 cells and because this serine protease is
predicted to possess tryptic-like enzymatic activity in
vivo, this protease probably plays an important convertase-like
role in the maturation of certain families of proteins.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1,
2,
I,
II,
III,
transmembrane tryptase
(TMT)1/tryptase
, and
eosinophil serine protease-1
(Esp-1)2 (1-7). The
corresponding complex of genes resides on the syntenic region of mouse
chromosome 17 at the interface between bands 17A3.3 and 17B1 (7-11).
Although our previous genomic blot analysis suggested the presence of
additional mouse tryptase genes in the family (7), the only genes
and/or transcripts cloned so far at the mouse complex are those that
encode mouse mast cell protease (mMCP) 6, mMCP-7, and mouse TMT (mTMT).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]dCTP using the Rediprime kit
(Amersham Pharmacia Biotech), it was hybridized to the RNA blot at
65 °C for 2 h in Quickhyb solution. The blot was washed twice
at room temperature for 20 min each in 2× SSC containing 0.1% SDS and
then twice at 65 °C for 20 min each in 0.2× SSC containing 0.1%
SDS before being exposed to BIOMAX film for 1-2 days. After the bound
mT4 probe was removed, the blot was reprobed with a
-actin cDNA
(CLONTECH) to evaluate the amount of RNA loaded in
each lane. In a similar manner, total RNA from normal mouse bone marrow
cells, IL-5 transgenic mouse bone marrow cells, and eosinophils
purified from IL-5 transgenic mice (21) were evaluated for the presence
of the mT4 transcript. Day-7, -11, -15, and -17 mouse embryos also were
evaluated for their mT4 mRNA levels.
-33P]dUTP (PerkinElmer Life Sciences) to generate
antisense and sense radiolabeled RNA probes. Tissue sections were
placed on slides, deparaffinized, fixed in 4% paraformaldehyde in PBS,
and treated with proteinase K. After washing in 0.5× SSC, the sections
were covered with 50 µl of hybridization solution (50% deionized
formamide, 0.3 M NaCl, 5 mM EDTA, 1×
Denhardt's solution, 10% dextran sulfate, 10 mM
dithiothreitol, and 20 mM Tris-HCl, pH 8.0) and incubated for 2 h at 55 °C. 33P-Labeled antisense or sense
RNA probes (3 million cpm/slide) were added to the hybridization
solution, and the sections were incubated for an additional 12-18 h at
55 °C. The resulting sections were washed for 20 min in 2× SSC, 10 mM
-mercaptoethanol, and 1 mM EDTA and then
were exposed to RNase A (10 µg/ml) for 30 min at room temperature. To
minimize the possibility of nonspecific binding of the radiolabeled
probe, each section was incubated for an additional 2 h at
60 °C in 0.1× SSC,
-mercaptoethanol, and 1 mM EDTA.
The resulting sections were dehydrated, dipped in photographic emulsion
NTB3 (Kodak), and stored at 4 °C. After 7 days of
exposure, the radiolabeled sections were developed, and cells in these
sections were counterstained with hematoxylin and eosin. In this
radioactive analysis, black silver granules appear over those cells
that contain abundant levels of mT4 mRNA.
-mercaptoethanol. After electrophoresis, the resolved
proteins were blotted onto polyvinylidene difluoride membranes
(Bio-Rad). Each protein blot was exposed to Tris-buffered saline (15 ml) containing 5% non-fat milk, 0.1% Tween 20, 0.5% goat serum, and
3 µg of mouse anti-V5 antibody (Invitrogen) for 2 h at room
temperature. After each blot was washed 3 times with Tris-buffered
saline containing 0.1% Tween 20, it was exposed to Tris-buffered
saline (15 ml) containing 5% non-fat milk, 0.1% Tween 20, 0.5% goat
serum, and a 1:1000 dilution of a stock solution of horseradish
peroxidase-conjugated goat anti-mouse IgG (Bio-Rad) for 1 h at
room temperature. The immunoreactive proteins were then visualized
using a chemiluminescence kit (Genotech, St. Louis, MO) and BioMax MR
film (Kodak). In order to confirm that mT4 is a membrane-anchored
protein, ~4 × 106 mT4-expressing COS-7 cells were
suspended in 750 µl of buffer (4 mM HEPES, pH 7.0, containing 50 mM sucrose, 0.4 mM EDTA, and 0.2 mM dithiothreitol) and lysed by subjecting the cell
suspension to multiple freeze-thaw cycles using liquid nitrogen and a
45 °C water bath. After centrifugation for 1 h at
>100,000 × g, aliquots of the obtained supernatant
representing the cytosolic fraction and aliquots of the pelleted
microsomal fraction were boiled in Laemmli buffer in the presence of
-mercaptoethanol and then subjected to SDS-PAGE/immunoblot analysis
as described above.
II (31). Within this region, the amino acid sequences
of the two tryptases are 39% identical.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (36K):
[in a new window]
Fig. 1.
Cloning of the mT4 cDNA.
A, a three-step approach was used to isolate the full-length
mT4 transcript. A computer search of GenBank's EST data base resulted
in the identification of an EST that encodes a portion of a novel mouse
serine protease. Based on its nucleotide sequence, 5'- and 3'-RACE
approaches were then carried out on a pool of mouse testis-derived
cDNAs to deduce the remaining portion of the expressed transcript.
The diagram in A highlights the 5'-UTR, prepropeptide, the
catalytic domain of the mature enzyme with its catalytic triad
(circled H, D, and S amino acid residues),
C-terminal hydrophobic segment, 3'-UTR, and poly(A) tail. B,
the nucleotide and amino acid sequences of the full-length mT4
transcript were deduced. The four potential N-linked
glycosylation sites in mT4 are circled in B and
the C-terminal hydrophobic extension is boxed. Components of
the catalytic triad ( ), translation-initiation site (*),
translation-termination site (
), leader peptide (single
bracket), and propeptide (double bracket) are
indicated. Nucleotide numbering begins at the 5'-UTR of the isolated
transcript; amino acid numbering (within brackets at left)
begins with the putative mature protein. C, a Kyte-Doolittle
hydropathy plot of the translated product was generated. The individual
residues in the coding region of mT4 are indicated on the x
axis; the extent of their hydrophobicity and hydrophilicity are on the
y axis.
View larger version (95K):
[in a new window]
Fig. 2.
Comparison of the amino acid sequences of mT4
with the most closely related known mouse serine proteases.
A, dendrogram comparing mT4 with the closely related serine
proteases in the mouse was generated by the GCG program "Distances"
using the unweighted pair group with arithmetic mean (UPGMA) algorithm.
(The mTesp-3 gene, transcript, and translated product
have not been described. Thus, the relationship of mT4 to this putative
testis-specific serine protease cannot be evaluated at this time.)
B, the amino acid sequences of hEsp-1, mTMT, mMCP-6, mMCP-7,
mAcrosin, mTesp-1, mTesp-2, mTesp-4, mHepsin, mKallikrein,
mTrypsin, hLeydin, and hProstasin were extracted from Swiss Protein
Database and aligned with the PILEUP program of the Eugene "GCG"
software package. Identical amino acids are shaded.
Numbering (left) begins at the first residue in the mature
portion of each protease. The C-terminal hydrophobic segment of mT4 is
bracketed. The seven putative loops (designated
A-D and 1-3) that form the substrate-binding
pocket of each of these proteases (also see Fig. 5B) are
underlined. The depicted amino acid sequence of mAcrosin is
truncated at the C terminus.
View larger version (77K):
[in a new window]
Fig. 3.
SDS-PAGE/immunoblot and
immunohistochemical analysis of mT4-expressing COS-7 cells.
A, COS-7 cells were transfected with expression vector alone
(left lanes) or expression vector containing an insert that
encodes a bioengineered form of mT4 possessing the immunogenic V5
peptide at its C terminus (right lanes). Forty eight hours
later, samples of the resulting conditioned media/supernatants
(S) and lysates of the cell pellets (P) were
analyzed for the presence of recombinant protein with anti-V5 antibody.
B, lysates of mT4-expressing COS-7 cells were fractionated
to determine whether or not mT4 is a membrane-anchored protein. Shown
is the immunoblot analysis of the membrane- (MF) and cytosol
(CF)-enriched fractions. C, a sample of the cell
lysates of mT4-expressing COS-7 cells was incubated 1 h in the
absence ( ) or presence (+) of PNGase F prior to SDS-PAGE/immunoblot
analysis to determine whether or not mT4 contains N-linked
glycans. Molecular mass markers are shown on the left. The
arrow and the open arrowhead on the
right point to glycosylated and nonglycosylated forms of
mT4, respectively. The deglycosylated product is ~35 kDa because it
contains the additional V5 and His6 peptides at its C
terminus. D-F, mT4-expressing cells were stained with a
mouse monoclonal antibody directed against the V5 peptide
(D), a mixture of rabbit antibodies directed against
calnexin and calreticulin (E), or antibodies directed
against all three epitopes (F). Yellow color in
F indicates co-localization of mT4 with calnexin and
calreticulin. Based on this double staining approach, a substantial
portion of the expressed mT4 is anchored in the ER membrane of the
cell.
View larger version (30K):
[in a new window]
Fig. 4.
Evaluation of the enzymatic activity of
insect cell-derived recombinant mT4. A, recombinant
mT4-FLAG was purified from the lysates of High Five insect cells using
an immunoaffinity chromatography approach. The soluble proteins in
lysates of mT4-FLAG-expressing cells were applied to an anti-FLAG
antibody column. After the column was washed extensively, the pH of the
buffer was changed to elute the bound recombinant protein.
B, fractions 5 and 6 of the immunoaffinity column were
pooled and evaluated for their enzymatic activity before ( ) and after
(+) enterokinase (EK) treatment. Trypsin and activating
buffer containing enterokinase alone were used as positive and negative
controls, respectively, in this casein-susceptibility assay. As
assessed by Coomassie Blue staining of a duplicate gel, the amount of
protein in fractions 5 and 6 was below detection. Thus, the amount of
trypsin used in the depicted experiment exceeds that of recombinant
mT4.
II, mMCP-6, and mMCP-7 to form tetramers. However,
mT4 possesses the functionally important surface Trp-rich domain found
in all other tryptases in this family. The presence of
Asp188, Gly215, and Gly225 in mT4
also strongly suggests that it is a tryptase. Nevertheless, the seven
loops that form its substrate-binding cleft are unique (Figs.
2B and 5B). For example, residue substitutions in
loops 3 and A in mT4 are predicted to result in shape differences
relative to human tryptase
II (Fig. 5B). Although mature
mT4 has an overall net
5 charge at neutral pH, it is predicted to
have two positively charged surface regions at diametrically opposite
ends of the folded protein (Fig. 5A). Arg33,
Arg34, and Arg243 reside in one region, whereas
His162, Lys165, Lys166,
Arg170, and Arg223 reside in the other
region.
View larger version (37K):
[in a new window]
Fig. 5.
Three-dimensional model of the catalytic
portion of mature mT4 based on the crystal structure of human
tryptase II. A, a
three-dimensional model of the catalytic domain of mature mT4 was
created. Shown is the overall structure of residues 1-256 of mT4.
Because human tryptase
II lacks a C-terminal hydrophobic domain, the
short membrane-spanning domain in mT4 was not modeled. The active site
residues (His41, Asp93, and Ser194)
are represented as green sticks. The free Cys residue 113 is
shown in orange. The side chains of the residues
(His162, Lys165, Lys166,
Arg170, Arg220, Arg223,
Arg33, Arg34, and Arg243) that
consist of the two positively charged surface regions are shown as
blue sticks. The C-
atoms of the conserved residues
(Trp12, Trp14, Trp128,
Trp206, Trp214, and Trp236) that
form the hydrophobic domain opposite the substrate-binding cleft are
shown as red spheres. Two of the conserved residues
(Trp35 and Trp174) in the domain are hidden in
this view. The figure was created with the programs Molscript (50) and
Raster3D (51). The general orientation of the mT4 model is similar to
that of the mMCP-6 (12), mMCP-7 (30), and mTMT (7) models in our
previous publications. B, the putative substrate-binding
cleft of mT4 was analyzed at a higher resolution using the modeling
approach. The 7 loops that form the substrate-binding cleft of mT4 are
marked A-D and 1-3 and are superimposed on the
corresponding loops of human tryptase
II. The loops in mT4 and human
tryptase
II are shown in red and blue,
respectively; the active site residues are shown as green
sticks.
View larger version (30K):
[in a new window]
Fig. 6.
Genomic blot analysis and chromosomal
location of the mT4 gene. A, a blot
containing mouse genomic DNA digested with EcoRI,
DraI, BamHI, AvrII, EcoRV,
BglII, or HindIII was probed under conditions of
high stringency with a radiolabeled 536-bp fragment derived from the 5'
end of the mT4 cDNA. DNA fragments of known molecular weight
(HindIII-digested DNA) are indicated on the
left of the blot. As noted in Fig. 7A, the
mT4 gene contains internal sites that are susceptible to
AvrII, HindIII, and BglII.
B, the chromosome location of the mT4 gene was
determined by FISH analysis. The fluorescent-labeled, mT4-containing
BAC clone hybridized specifically to a small-sized chromosome
(arrow, left panel) that was subsequently shown
to be chromosome 17. The location (arrow) of the
mT4 gene on this chromosome is more clearly indicated in the
right panel.
View larger version (90K):
[in a new window]
Fig. 7.
Structure of the mT4
gene. A, the nucleotides that consist of the six
exons and five introns of the mT4 gene were deduced and are
shown in upper and lowercase letters,
respectively. The exons are boxed, and the deduced amino
acid sequence of the translated product is indicated, as well as the
components of the catalytic triad ( ). The putative polyadenylation
signal site in exon 6 is underlined. The BglII
and AvrII restriction sites in introns 3 are
italicized. B, the exon/intron organization of the
mT4 gene was compared with that of the mTMT,
mMCP-6, and mMCP-7 genes. Boxes (
)
indicate exons. The size of each gene is indicated on the right.
H, D, and S refer to the catalytic triad amino acids in
each tryptase.
View larger version (24K):
[in a new window]
Fig. 8.
Distribution of the mT4 transcript in tissues
and cells. A, a blot containing total RNA from normal
mouse bone marrow (lane 2), bone marrow from IL-5 transgenic
mice (lane 1), and eosinophils purified from IL-5 transgenic
mice (lane 3) was probed under conditions of high stringency
with the mT4 cDNA (upper panel). The amount of 28 S
ribosomal RNA in each lane is shown in the lower panel. Much
less total RNA was intentionally loaded in lane 3 in order
to show that the eosinophils are the major cell type in the bone marrow
of the IL-5 transgenic mice that expresses mT4. B, RT-PCR
was used to evaluate the levels of mT4 mRNA in the liver, spleen,
and intestine of a day-7-infected mouse and a day-14-infected mouse.
The arrow (left) points to the ~280-bp product
that was generated only from the eosinophil-enriched intestine of the
day-7-infected animal. The positive (testis RNA) and negative (no RNA)
controls used in this RT-PCR are shown. Control RT-PCRs also were
carried out with -actin-specific primers to confirm that each sample
contained non-degraded mRNA (data not shown). C, a blot
containing poly(A)+ RNA from various mouse tissues was
probed under conditions of high stringency with the mT4 cDNA
(upper panel). The blot was then reprobed with the
-actin
cDNA (lower panel) to demonstrate that comparable
amounts of RNA are present in each lane. Molecular mass markers are
indicated on the left. D, RT-PCR analysis
was carried out on RNA samples isolated from the testis and ovaries of
3- and 8-week-old mice. RNA blot analysis (data not shown) confirmed
the presence of mT4 in the testes and ovaries of 8-week-old mice.
View larger version (118K):
[in a new window]
Fig. 9.
Location of mT4-expressing cells in the
testis by in situ hybridization. To identify the
cell type(s) in the testis that contains abundant levels of mT4
mRNA, a JB4 glycomethacrylate-embedded mouse testis was sectioned
and probed with an mT4-specific, digoxigenin-labeled oligonucleotide
(A and C) using an in situ
hybridization approach. For a negative control (B and
D), the reaction was carried out on a replicate slide in the
absence of the labeled oligonucleotide. Shown are different
magnifications of the resulting tissue sections. Based on this
analysis, the mT4 transcript (arrows, A and C) is
transiently expressed relatively late in spermatogenesis. This
conclusion was confirmed using a radioactive in situ
hybridization approach with antisense (E) and sense
(F) mT4 RNA probes.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
II (31), Cys26,
Cys42, Cys127, Cys160,
Cys179, Cys190, Cys200, and
Cys218 are predicted to form 4 disulfide bonds in the
catalytic portion of the mature, properly folded protease (Fig.
5A). One of the additional Cys resides at residue
9 in the
propeptide; the other resides at residue 113. Although
Cys113 is not present in mMCP-6 or mMCP-7 (Fig.
2B), a corresponding Cys is present in the two-chained
proteases factor XI (38), plasma kallikrein (39), and acrosin (40).
Because this Cys forms a disulfide bond with a Cys residue in the
propeptide of each zymogen, mature mT4 could be a two-chain serine
protease consisting of a 33-residue, non-catalytic N-terminal chain
covalently linked to the larger sized catalytic C-terminal chain.
II (31), Gly225 is conserved because it
contacts Asp188 at the base of the substrate-binding cleft.
Gly215 resides in loop 2 (Fig. 5), and this surface loop
helps define the substrate specificity of the serine protease (42).
Although human tryptase
I is an exception (25), tryptases have a Gly at the corresponding site presumably because this small-sized amino
acid residue facilitates entry of the bulky P1 residue of the substrate
into the pocket of the enzyme.
![]() |
ACKNOWLEDGEMENT |
---|
We thank Carmen Tam (In Situ Core Facility, Dana Farber Cancer Institute, Boston, MA) for technical assistance in the radioactive in situ hybridization analysis of mT4-expressing cells in the testis.
![]() |
FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grants AI-23483, HL-36110, and HL-63284 and by a grant from the Mizutani Foundation for Glycoscience.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF176209 and AF226710.
§ Pharmacia Allergy Research Foundation Fellow.
Alfred P. Sloan Research Fellow.
§§ To whom correspondence and reprint requests should be addressed: Brigham and Women's Hospital, Dept. of Medicine, Smith Bldg., Rm. 616B, 1 Jimmy Fund Way, Boston, MA 02115. Tel.: 617-525-1231; Fax: 617-525-1310; E-mail: rstevens@rics.bwh.harvard.edu.
Published, JBC Papers in Press, March 20, 2001, DOI 10.1074/jbc.M010422200
2 Genomic sequencing of the 35.7-kb cosmid clone 352F10 by D. O. Ricke and co-workers (GenBankTM direct submission, accession number AC005361) resulted in the initial identification and chromosomal location of the 4.6-kb human gene in 1998 that others (4, 5, 44) designated as the hEsp-1/testisin gene in 1999.
3 R. L. Stevens, unpublished observations.
4 G. W. Wong and R. L. Stevens, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: TMT, transmembrane tryptase; ER, endoplasmic reticulum; hEsp-1, human eosinophil serine protease-1; FISH, fluorescent in situ hybridization; MC, mast cell; mMCP, mouse MC protease; mT4, mouse tryptase 4; PBS, phosphate-buffered saline; RACE, rapid amplification of cDNA ends; mTesp, mouse testicular serine protease; UTR, untranslated region; EST, expressed sequence tag; kb, kilobase pairs; bp, base pairs; PCR, polymerase chain reaction; RT-PCR, reverse transcriptase-PCR; IL, interleukin; PAGE, polyacrylamide gel electrophoresis; PNGase F, peptide N-glycosidase F.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Miller, J. S., Westin, E. H., and Schwartz, L. B. (1989) J. Clin. Invest. 84, 1188-1195[Medline] [Order article via Infotrieve] |
2. | Miller, J. S., Moxley, G., and Schwartz, L. B. (1990) J. Clin. Invest. 86, 864-870[Medline] [Order article via Infotrieve] |
3. | Vanderslice, P., Ballinger, S. M., Tam, E. K., Goldstein, S. M., Craik, C. S., and Caughey, G. H. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 3811-3815[Abstract] |
4. | Inoue, M., Kanbe, N., Kurosawa, M., and Kido, H. (1998) Biochem. Biophys. Res. Commun. 252, 307-312[CrossRef][Medline] [Order article via Infotrieve] |
5. | Inoue, M., Isobe, M., Itoyama, T., and Kido, H. (1999) Biochem. Biophys. Res. Commun. 266, 564-568[CrossRef][Medline] [Order article via Infotrieve] |
6. |
Pallaoro, M.,
Fejzo, M. S.,
Shayesteh, L.,
Blount, J. L.,
and Caughey, G. H.
(1999)
J. Biol. Chem.
274,
3355-3362 |
7. |
Wong, G. W.,
Tang, Y.,
Feyfant, E.,
![]() |
8. |
Gurish, M. F.,
Nadeau, J. H.,
Johnson, K. R.,
McNeil, H. P.,
Grattan, K. M.,
Austen, K. F.,
and Stevens, R. L.
(1993)
J. Biol. Chem.
268,
11372-11379 |
9. | Gurish, M. F., Johnson, K. R., Webster, M. J., Stevens, R. L., and Nadeau, J. H. (1994) Mamm. Genome 5, 656-657[Medline] [Order article via Infotrieve] |
10. | McNeil, H. P., Reynolds, D. S., Schiller, V., Ghildyal, N., Gurley, D. S., Austen, K. F., and Stevens, R. L. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 11174-11178[Abstract] |
11. |
Reynolds, D. S.,
Gurley, D. S.,
Austen, K. F.,
and Serafin, W. E.
(1991)
J. Biol. Chem.
266,
3847-3853 |
12. |
Ghildyal, N.,
Friend, D. S.,
Stevens, R. L.,
Austen, K. F.,
Huang, C.,
Penrose, J. F.,
![]() |
13. | Stevens, R. L., Friend, D. S., McNeil, H. P., Schiller, V., Ghildyal, N., and Austen, K. F. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 128-132[Abstract] |
14. |
Huang, C.,
Wong, G. W.,
Ghildyal, N.,
Gurish, M. F.,
![]() |
15. | Echtenacher, B., Männel, D. N., and Hültner, L. (1996) Nature 381, 75-77[CrossRef][Medline] [Order article via Infotrieve] |
16. | Malaviya, R., Ikeda, T., Ross, E., and Abraham, S. N. (1996) Nature 381, 77-80[CrossRef][Medline] [Order article via Infotrieve] |
17. | Prodeus, A. P., Zhou, X., Maurer, M., Galli, S. J., and Carroll, M. C. (1997) Nature 390, 172-175[CrossRef][Medline] [Order article via Infotrieve] |
18. | Reynolds, D. S., Stevens, R. L., Lane, W. S., Carr, M. H., Austen, K. F., and Serafin, W. E. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 3230-3234[Abstract] |
19. | Johnson, A. R., Hugli, T. E., and Müller-Eberhard, H. J. (1975) Immunology 28, 1067-1080[Medline] [Order article via Infotrieve] |
20. |
Huang, C.,
Friend, D. S.,
Qiu, W. T.,
Wong, G. W.,
Morales, G.,
Hunt, J.,
and Stevens, R. L.
(1998)
J. Immunol.
160,
1910-1919 |
21. | Dent, L. A., Strath, M., Mellor, A. L., and Sanderson, C. J. (1990) J. Exp. Med. 172, 1425-1431[Abstract] |
22. | Friend, D. S., Ghildyal, N., Austen, K. F., Gurish, M. F., Matsumoto, R., and Stevens, R. L. (1996) J. Cell Biol. 135, 279-290[Abstract] |
23. |
Benyon, R. C.,
Enciso, J. A.,
and Befus, A. D.
(1993)
J. Immunol.
151,
2699-2706 |
24. |
Ghildyal, N.,
Friend, D. S.,
Freelund, R.,
Austen, K. F.,
McNeil, H. P.,
Schiller, V.,
and Stevens, R. L.
(1994)
J. Immunol.
153,
2624-2630 |
25. |
Huang, C.,
Li, L.,
Krilis, S. A.,
Chanasyk, K.,
Tang, Y.,
Li, Z.,
Hunt, J. E.,
and Stevens, R. L.
(1999)
J. Biol. Chem.
274,
19670-19676 |
26. |
Huang, C.,
Morales, G.,
Vagi, A.,
Chanasyk, K.,
Ferrazzi, M.,
Burklow, C.,
Qiu, W. T.,
Feyfant, E.,
![]() |
27. |
![]() |
28. |
![]() |
29. |
![]() |
30. |
Matsumoto, R.,
![]() |
31. | Pereira, P. J., Bergner, A., Macedo-Ribeiro, S., Huber, R., Matschiner, G., Fritz, H., Sommerhoff, C. P., and Bode, W. (1998) Nature 392, 306-311[CrossRef][Medline] [Order article via Infotrieve] |
32. | Kozak, M. (1989) J. Cell Biol. 108, 229-241[Abstract] |
33. | Breathnach, R., and Chambon, P. (1981) Annu. Rev. Biochem. 50, 349-383[CrossRef][Medline] [Order article via Infotrieve] |
34. |
Friend, D. S.,
Gurish, M. F.,
Austen, K. F.,
Hunt, J.,
and Stevens, R. L.
(2000)
J. Immunol.
165,
344-352 |
35. |
Wei, L.,
Alhenc-Gelas, F.,
Soubrier, F.,
Michaud, A.,
Corvol, P.,
and Clauser, E.
(1991)
J. Biol. Chem.
266,
5540-5546 |
36. |
Yu, J. X.,
Chao, L.,
and Chao, J.
(1995)
J. Biol. Chem.
270,
13483-13489 |
37. | Baba, T., Watanabe, K., Kashiwabara, S., and Arai, Y. (1989) FEBS Lett. 244, 296-300[CrossRef][Medline] [Order article via Infotrieve] |
38. | McMullen, B. A., Fujikawa, K., and Davie, E. W. (1991) Biochemistry 30, 2056-2060[Medline] [Order article via Infotrieve] |
39. | McMullen, B. A., Fujikawa, K., and Davie, E. W. (1991) Biochemistry 30, 2050-2056[Medline] [Order article via Infotrieve] |
40. | Topfer-Petersen, E., Calvete, J., Schafer, W., and Henschen, A. (1990) FEBS Lett. 275, 139-142[CrossRef][Medline] [Order article via Infotrieve] |
41. | Ruhlmann, A., Kukla, D., Schwager, P., Bartels, K., and Huber, R. (1973) J. Mol. Biol. 77, 417-436[Medline] [Order article via Infotrieve] |
42. | Perona, J. J., Tsu, C. A., Craik, C. S., and Fletterick, R. J. (1993) J. Mol. Biol. 230, 919-933[CrossRef][Medline] [Order article via Infotrieve] |
43. |
Johnson, D. A.,
and Barton, G. J.
(1992)
Protein Sci.
1,
370-377 |
44. |
Hooper, J. D.,
Nicol, D. L.,
Dickinson, J. L.,
Eyre, H. J.,
Scarman, A. L.,
Normyle, J. F.,
Stuttgen, M. A.,
Douglas, M. L.,
Loveland, K. A.,
Sutherland, G. R.,
and Antalis, T. M.
(1999)
Cancer Res.
59,
3199-3205 |
45. | Saling, P. M. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 6231-6235[Abstract] |
46. |
Baba, T.,
Azuma, S.,
Kashiwabara, S.,
and Toyoda, Y.
(1994)
J. Biol. Chem.
269,
31845-31849 |
47. | Kohno, N., Yamagata, K., Yamada, S., Kashiwabara, S., Sakai, Y., and Baba, T. (1998) Biochem. Biophys. Res. Commun. 245, 658-665[CrossRef][Medline] [Order article via Infotrieve] |
48. |
Ohmura, K.,
Kohno, N.,
Kobayashi, Y.,
Yamagata, K.,
Sato, S.,
Kashiwabara, S.,
and Baba, T.
(1999)
J. Biol. Chem.
274,
29426-29432 |
49. |
Steiner, D. F.,
Smeekens, S. P.,
Ohagi, S.,
and Chan, S. J.
(1992)
J. Biol. Chem.
267,
23435-23438 |
50. | Kraulis, P. J. (1991) J. Appl. Crystallogr. 24, 946-950[CrossRef] |
51. | Merritt, E. A., and Murphy, M. E. P. (1994) Acta Crystallogr. Sect. D Biol. Crystallogr. 50, 869-873[CrossRef][Medline] [Order article via Infotrieve] |