J Biol Chem, Vol. 274, Issue 43, 30784-30793, October 22, 1999
Identification of a New Member of the Tryptase Family of
Mouse and Human Mast Cell Proteases Which Possesses a Novel
COOH-terminal Hydrophobic Extension*
Guang W.
Wong
,
Yinzi
Tang,
Eric
Feyfant§,
Andrej
ali§¶,
Lixin
Li
,
Yong
Li,
Chifu
Huang,
Daniel S.
Friend**,
Steven A.
Krilis, and
Richard L.
Stevens
From the Departments of Medicine and ** Pathology,
Harvard Medical School, and Brigham and Women's Hospital, Boston,
Massachusetts 02115, the § Rockefeller University, New York,
New York 10021, and the Department of Immunology, Allergy, and
Infectious Disease, St. George Hospital, and the University of New
South Wales, Kogarah, New South Wales, 2217 Australia
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ABSTRACT |
Mapping of the tryptase locus on chromosome 17 revealed a novel gene 2.3 kilobase 3' of the mouse mast cell
protease (mMCP) 6 gene. This 3.7-kilobase
gene encodes the first example of a protease in the tryptase family
that contains a membrane-spanning segment located at its COOH terminus.
Comparative structural studies indicated that the putative
transmembrane tryptase (TMT) possesses a unique substrate-binding
cleft. As assessed by RNA blot analyses, mTMT is expressed
in mice in both strain- and tissue-dependent manners. Thus,
different transcriptional and/or post-transcriptional mechanisms are
used to control the expression of mTMT in vivo. Analysis of
the corresponding tryptase locus in the human genome resulted in the
isolation and characterization of the hTMT gene. The
hTMT transcript is expressed in numerous tissues and is
also translated. Analysis of the tryptase family of genes in mice and humans now indicates that a primordial serine protease gene duplicated early and often during the evolution of mammals to generate a panel of
homologous tryptases in each species that differ in their tissue
expression, substrate specificities, and physical properties.
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INTRODUCTION |
Tryptases are stored in abundance in the secretory granules of
mouse (1-4), rat (5-7), gerbil (8), dog (9), and human (10-15) mast
cells (MCs).1 In humans, the
four homologous tryptases (designated tryptases I, II/
,
III, and 
) that have been cloned reside at a complex on
chromosome 16 (16). Although only two tryptases (designated mouse
MC protease (mMCP) 6 and mMCP-7)
have been identified so far in the mouse, their genes reside ~1.2
centimorgans away from each other on the syntenic region of mouse
chromosome 17 (17, 18). Despite the chromosomal clustering of their
genes, these mouse tryptases are differentially regulated in
vivo (1, 19-21) and in vitro (2, 3) at the levels of
gene transcription (22) and mRNA
stability.2
All known mouse and human tryptases in this family are initially
translated as zymogens. They possess an ~20-residue hydrophobic signal peptide which is presumed to be removed in the endoplasmic reticulum immediately after the translated zymogen is translocated into
the lumen. They also possess an ~10-residue propeptide preceding the
mature portion of the enzyme which consists of ~245 amino acids. No
mature tryptase with a membrane-spanning segment in its COOH terminus
has been found in any species so far. Although tryptases undergo
variable N-linked glycosylation during their biosynthesis
(5, 12, 23, 24), the current members of the family appear to be
targeted to the secretory granule by a serglycin
proteoglycan-dependent mechanism (25, 26) rather than by a
Man-PO4-dependent mechanism as are classical
lysosomal enzymes.
The amino acid sequences of mMCP-6 and mMCP-7 are 71% identical
(1-4). Nevertheless, these homologous tryptases have different physicochemical properties (25, 26) and substrate preferences (27-29).
Recent in vivo studies have suggested that mMCP-6 and mMCP-7
evolved to carry out different functions. In mice, mMCP-6 regulates
neutrophil extravasation into tissues (28, 29), whereas mMCP-7 helps to
minimize the deposition of fibrin/platelet thrombi during a MC-mediated
inflammatory reaction (27).
The findings that recombinant mMCP-6 and mMCP-7 exhibit potent but
different bioactivities in vivo highlight the need to
identify and characterize all of the tryptase genes present in the
mouse and human genomes. Because of the importance of mouse tryptases in inflammation (27-29), because more tryptases have been cloned from
the human genome (13-16) than from the mouse genome (2, 3), and
because adjacent serine protease genes in large superfamilies often
reside within 7 kb of one another on their respective chromosomes (30,
31), a walk approach was carried out to identify the functional gene
that resides on chromosome 17 immediately 3' of the mMCP-6
gene. We now describe a novel mouse gene, and its human ortholog, which
encode an unusual transmembrane tryptase (TMT).
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EXPERIMENTAL PROCEDURES |
Cloning and Sequencing of the mTMT Gene--
A 
bacteriophage
clone (designated GW-1) was isolated by screening a 129/Sv mouse
genomic library (Stratagene, La Jolla, CA) under conditions of high
stringency with a radiolabeled probe specific for the mMCP-6
gene (2). A 6-kb BamHI-derived fragment liberated from GW-1
was subcloned into pBluescript (Stratagene) and its nucleotide sequence
determined in both directions with standard dideoxy/cycle sequencing
methodologies (32). Analysis of the obtained nucleotide sequence data
revealed that this portion of chromosome 17 contained exon 6 of the
mMCP-6 gene, followed by 2.3 kb of flanking DNA, and then
the 3.7-kb mTMT gene. The mMCP-6 and
mTMT genes were oriented in the same direction in GW-1. To
confirm the position and spacing of the two genes in native chromosomal
DNA, a long range polymerase chain reaction (PCR) approach was carried
out in which each 50-µl sample contained 200 ng of BALB/c or 129/Sv
mouse genomic DNA, 10 µmol of an oligonucleotide (5'-ACTCACTGCTTCCTGGTCAG-3') that corresponds to a region in exon 6 of
the mMCP-6 gene, and 10 µmol of an oligonucleotide
(5'-ACAGTGTGACCGTAAGCTC-3') that corresponds to a region in exon 3 of
the mTMT gene. Thirty cycles of PCR were performed with
recombinant Thermus thermophilius-derived DNA polymerase
(Perkin-Elmer); each cycle consisted of a 30-s denaturing step at
94 °C, a 30-s annealing step at 58 °C, and a 4-min extension step
at 72 °C. The amplified PCR products were subcloned into the TA
vector pCR 2.1 (Invitrogen, San Diego, CA) and subjected to nucleotide
sequence analysis.
To determine if homologous mTMT genes exist in the mouse
genome, ~20-µg samples of BALB/c mouse genomic DNA were incubated separately at 37 °C for ~17 h with BamHI,
ScaI, HindIII, PstI, BglII,
SacI, AvrII, or XbaI (New England
Biolabs, Beverly, MA). The digests were fractionated on 1% agarose
gels. The separated fragments were blotted onto MagnaGraph nylon
membranes (Micron Separations Inc., Westborough, MA) (33), and the
resulting DNA blots were incubated for 2 h at 65 °C in QuikHyb
hybridization solution (Stratagene) containing a radiolabeled 224-base
pair (bp) probe corresponding to a portion of exon 3 of the
mTMT gene. This probe was chosen because it corresponds to a
region in the mTMT transcript that is not present in the
mMCP-6 and mMCP-7 transcripts. The DNA blots were
washed twice for 15 min each at room temperature in 2 × SSC
containing 0.1% SDS and then twice for 15 min each either at 65 °C
or 50 °C in 0.2 × SSC containing 0.1% SDS. The blots were
then exposed to BIOMAX film for ~3 days. In some instances, the DNA
blots were stripped and reprobed with mMCP-6 (2) and mMCP-7 (3) gene-specific probes.
Isolation and Characterization of the mTMT Transcript, and
Evaluation of Its Expression in Different Tissues and in mBMMCs
Developed from Different Strains--
Total RNA was isolated (34) from
the v-abl-transformed V3 mouse MC line (21), non-transformed
MCs (mBMMCs) developed with interleukin 3 (35) from the bone marrows of
W/Wv (also know as KitW/KitW-v),
BALB/c, C57BL/6, and 129/Sv mice, and from varied tissues of BALB/c and
C57BL/6 mice. The RNA samples were applied to individual lanes of 1.2%
agarose-formaldehyde gels (36). The gels were subjected to
electrophoresis for 17 h, the separated RNA was transferred to
nylon membranes (Schleicher and Schuell), and the resulting blots were
analyzed with gene-specific probes for the mTMT, mMCP-6 (2),
mMCP-7 (3), and -actin (37) transcripts. The
cDNA probes used in these analyses were random primed with
[-32P]dCTP using the rediprime kit
(Amersham Pharmacia Biotech) and hybridized to the RNA blots at
65 °C for 2 h in QuikHyb hybridization solution (Stratagene).
The blots were washed twice for 15 min each at room temperature in
2 × SSC containing 0.1% SDS, and then twice for 10 min each at
60 °C in 0.2 × SSC containing 0.1% SDS before exposure to film.
Three different approaches were used to demonstrate that the newly
identified mouse gene was transcribed in vitro and in
vivo in different mouse strains. A search of the GenBank mouse
expressed sequence tag (EST) data base revealed that clone AA266560
probably corresponded to a portion of the mTMT gene. This
EST was derived from mixed organs of a FVB/N mouse. Sequence analysis
of the EST, obtained from the "Integrated Molecular Analysis of
Genomes and their Expression" (I.M.A.G.E.) consortium, revealed that
it corresponded to residues 109 to 1112 in the 1.2-kb mTMT
transcript. To obtain the 5' portion of the mTMT transcript,
5'-Marathon RACE (rapid amplification of cDNA ends) was performed
on a CLONTECH (Palo Alto, CA) preparation of BALB/c
mouse liver-derived cDNAs, according to the manufacture's
instructions. The first PCR was carried out using the anchor
oligonucleotide 5'-CCATCCTAATACGACTCACTATAGGGC-3' and the
oligonucleotide 5'-ATCCACCACAGAGACTTTGGCCTCCTGAGG-3' which corresponds
to a region in exon 4 of the mTMT gene. The second nested
PCR was carried out using the second anchor oligonucleotide 5'-ACTCACTATAGGGCTCGAGCGGC-3' and the oligonucleotide
5'-CATCCCAGGGTAGAAGTCAGCTGAGGCCTC-3' which corresponds to a region in
exon 3 of the mTMT gene. Amplified products were subcloned
into pCR 2.1 (Invitrogen) and their inserts sequenced. A reverse
transcription (RT) PCR approach was then used to confirm that the
mTMT transcript is expressed in W/Wv mBMMCs. Two
sets of oligonucleotides were used in these latter reactions. The first
(5'-CAGGCTAGCCTCCGTCTG-3' and 5'-CATCCCAGGGTAGAAGTCAGC-3') and second
(5'-CTGTGAACTCGTCTGATTATC-3' and
5'-ACACCTCATTCAGAGTTCCGAGGCCGCGTG-3') sets of
oligonucleotides cover exons 2 to 3 and exons 3 to 5, respectively, in
the mTMT gene. The RT step was carried out at 55 °C for
30 min with a kit from Roche Molecular Biochemicals (Indianapolis, IN).
Each of the 30 cycles of the PCR consisted of a 15-s denaturing step at
94 °C, a 30-s annealing step at 58 °C, and a 60-s extension step
at 72 °C.
Isolation and Characterization of the hTMT Transcript and Gene,
and Chromosomal Location of the hTMT Gene--
A PCR approach was used
to determine whether or not there is a human ortholog of the
mTMT transcript. A number of relatively conserved regions
were found when the nucleotide sequence of the mTMT
transcript was compared with those of varied mouse and human MC
tryptase transcripts. Thus, an oligonucleotide
(5'-TGCTGGGTCACTGGCTGG-3') that corresponds to a relatively conserved
region in all mouse and human MC tryptase transcripts and an
oligonucleotide (5'-GATCCAGTTCACGTAGGC-3') that corresponds to the 3'
end of the mTMT transcript were employed to amplify from a
pool of human liver cDNAs (CLONTECH) a 326-bp fragment that corresponds to the middle portion of the hTMT
transcript. Based on the nucleotide sequence of this PCR product, more
specific oligonucleotides were used in 5'- and 3'-RACE approaches to
obtain a more full-length hTMT transcript. In the case of
5'-RACE, the oligonucleotides 5'-CCAGCTCACAATGCCAGCCTGCACCCAG-3' and
5'-GCATGTCGGGCTGAAGGATGCTGC-3' were employed in the first and second
nested PCRs, respectively, with the relevant anchor oligonucleotides.
In the case of 3'-RACE, the oligonucleotide
5'-CGTACAGCCTGCGGGAGGTGAAAGTCTC-3' was used with the anchor
oligonucleotide. Reactions were performed with human liver and uterus
Marathon cDNAs (CLONTECH) as templates. Using
the primers 5'-AGGTGCACCTGGGGGAACTGGAGATCAC-3' and
5'-AATGCACTTGGATTCCTGCCATCAGTCAG-3', PCRs were also performed with
human skin (Invitrogen) and cecum cDNA libraries. All amplified PCR
products were subcloned into pCR 2.1 and their inserts sequenced.
A nested PCR approach was then used to elucidate which human adult,
fetal, and tumor tissues contained hTMT mRNA. The
oligonucleotides 5'-AGGTGCACCTGGGGGAACTGGAGATCAC-3' and
5'-AATGCACTTGGATTCCTGCCATCAGTCAG-3' and then the oligonucleotides
5'-ACCGTGAGGCAGATCATCCTGCACTCCAG-3' and
5'-CCAGCTCACAATGCCAGCCTGCACCCAG-3' were used in this two-step process
to generate the relevant 410-bp hTMT cDNA from four
different human tissue cDNA panels (CLONTECH).
The obtained products were analyzed by gel electrophoresis.
Glyceraldehyde-3-phosphate dehydrogenase oligonucleotides
(CLONTECH) were used as a positive control in these
mRNA analyses.
Based on the nucleotide sequence of the isolated hTMT
transcript, four sets of oligonucleotides were exploited to amplify its
gene in three overlapping fragments from human genomic DNA. The
oligonucleotides 5'-CCGGTGTGTCCCTCAGGACTTTGCAG-3' and
5'-CGCCGCACACGTGCATCCTCCGCAG-3' and the oligonucleotides
5'-AGGTGCACCTGGGGGAACTGGAGATCAC-3' and 5'-AATGCACTTGGATTCCTGCCATCAGTCAG-3' were used to isolate the
portions of the hTMT gene that span exon 1 to exon 2 and
exon 3 to exon 5, respectively. A nested PCR approach with
oligonucleotides 5'-GCGCATGGCCATGGCAG-3' and
5'-CCAGCTCACAATGCCAGCCTGCACCCAG-3', followed by the oligonucleotides 5'-GCAGGCCAGCCTCCGC-3' and 5'-GCATGTCG- GGCTGAAGGATGCTGC-3 were used to isolate the portion of the hTMT gene which spans
exon 2 to exon 4.
A panel of 24 hamster/human somatic hybrid cell lines (Quantum
Biotechnologies, Montreal, Canada) was probed to locate the TMT gene in the human genome. Each analyzed hybrid cell line
contained a single, but different, human chromosome. Approximately 200 ng of genomic DNA from a cell line was utilized as the template in each
50-µl PCR. Normal human genomic DNA and normal hamster genomic DNA
served as positive and negative controls, respectively. The sense and
antisense oligonucleotides in these PCRs were
5'-CGTACAGCCTGCGGGAGGTGAAAGTCTC-3' and 5-TAATCTGATGCAGAAGACTCAGC-3',
respectively. The obtained products were analyzed by gel
electrophoresis and then sequenced.
Immunohistochemistry--
The anti-peptide approach used to
obtain mMCP-6- (26) and mMCP-7- (24) specific antibodies in rabbits was
employed to obtain hTMT-specific antibodies. The 16-mer peptide
Val-Pro-Ala-Tyr-Val-Asn-Trp-Ile-Arg-Arg-His-Ile-Thr-Ala-Ser-Gly, which
corresponds to residues 221 to 236 in mature hTMT, is not present in
any protein in the GenBank protein data base. The models of the
three-dimensional structures3
of hTMT and mTMT predicted that this peptide would protrude from the
surface of the folded tryptase. Thus, antibodies were raised in rabbits
against the synthetic peptides by Quality Controlled Biochemicals
(Hopkinton, MA); the synthetic peptide was then used to affinity purify
the antibodies.
Immunohistochemistry was carried out essentially as described for other
anti-tryptase antibodies (24, 26). The anti-hTMT antibodies were used
to evaluate hTMT expression in human skin and large intestine. For
these experiments, histologically normal skin (n = 3)
or intestine (n = 2) specimens were snap frozen and placed in Tissue-TekTM O.C.T. compound (Sakura
Finetechnical Co., Tokyo, Japan). Cryostat sections (5 µm) were cut
in a Reichert-Jung cryostat, mounted on gelatin-coated glass slides,
and air-dried overnight at room temperature. Each section was fixed for
20 min at room temperature in 4% paraformaldehyde/phosphate-buffered
saline. After the sections were incubated for 3 h at 37 °C with
a 1:50 dilution of anti-hTMT immunoglobulin (Ig) in phosphate-buffered
saline, they were sequentially washed in phosphate-buffered saline,
incubated for 1 h with biotinylated anti-rabbit IgG
F(ab')2 (3.6 µg/ml) (Dakopatts, Glostrup, Denmark), for
1 h with alkaline phosphatase-conjugated streptavidin (Silinus, Hawthorn, Australia), and for 20 min with alkaline phosphatase substrate (0.2 mg/ml solution of naphthol 3-hydroxy-2-naphthonic acid
2,4-dimethylanilide phosphate (Sigma) containing 0.1 mg/ml Fast Red
4-chloro-2-methylbenzenediazonium (Sigma) in 0.1 M
Tris-HCl, pH 8.2).
Human skin biopsies were also processed for immunoelectron microscopy
in order to determine where TMT resides inside human cutaneous MCs.
Tissue blocks (2 mm3) were immersed for 4 h at room
temperature in a 0.1 M cacodylate buffer, pH 7.4, containing 1.25% glutaraldehyde, 1% paraformaldehyde, and 0.025%
CaCl2. After an overnight incubation at 4 °C in 0.1 M cacodylate buffer, the blocks were postfixed for 2 h
at room temperature in 2% osmium tetroxide. They were then stained for 2 h at room temperature with 2% of uranyl acetate, dehydrated in
graded ethanol, and embedded in Spurr's low viscosity media (ProSciTech, Thuringowa, Australia). Sections (60-80 nm) were cut on
an Reichert-Jung ultramicrotome and placed on grids. The grids were
placed upon drops of reagents on the parafilm, etched for 15 min in
10% of hydrogen peroxide, and washed with water. Each section was
equilibrated with Tris-HCl, 1% bovine serum albumin, pH 8.2, before
the 4-h incubation at 37 °C with primary antibody diluted in this
buffer. Antibody-treated sections were washed with the Tris-HCl/albumin
buffer and then exposed for 1 h at 37 °C to gold-labeled
anti-rabbit IgG (dilution of 1:50) (Ted Pella Inc., Redding, CA). The
resulting sections were counterstained with lead citrate before being
examined with a Hitachi 7000 electron microscope. For a negative
control, sections were not exposed to the primary antibody.
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RESULTS |
Cloning and Analysis of the mTMT Gene--
Mapping analysis with
varied restriction enzymes revealed that the phage clone GW-1
contained two homologous but distinct genes in its ~13-kb insert
(Fig. 1A). These genes were
oriented in the same direction in GW-1 and were separated from one
another by only 2.3 kb of flanking DNA. Nucleotide sequencing analysis revealed that one of the genes encoded mMCP-6; the other encoded a
novel tryptase. A long-range PCR approach (Fig. 1B), carried out with both BALB/c and 129/Sv mouse genomic DNA, confirmed the closeness of the two genes on chromosome 17. The novel mTMT
gene in GW-1 was 3.7 kb in size and consisted of 5 exons. The
nucleotide sequence of the mTMT gene and its exon/intron
organization are shown in Fig. 2, as well
as the nucleotide sequence that separates the mMCP-6 and
mTMT genes.
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Fig. 1.
Organization of the mMCP-6 and mTMT genes on chromosome 17. A, the depicted map of the phage genomic clone GW-1 is
not drawn to scale. Nevertheless, the six exons of the
mMCP-6 gene and the five exons of the mTMT gene
are boxed and numbered. The two genes are
separated by 2.3 kb of flanking DNA. B, long range PCRs were
performed with genomic DNA from BALB/c (lane 2) and 129/Sv
mice (lane 3). The arrows ( ) in
A indicate the locations of the two chromosome 17-derived
oligonucleotides used in these reactions. The arrow on the
right in B, indicates the generated 5-kb PCR
product that spans from exon 6 of the mMCP-6 gene to exon 3 of the mTMT gene. A 1-kb DNA ladder (Life Technologies,
Inc., Grand Island, NY) was used in lane 1 to determine the
molecular weights of the generated PCR products. An identical sized
fragment was generated when a similar long range PCR was performed on
GW-1 (data not shown). The PCR products were subcloned and partially
sequenced to confirm that they corresponded to the appropriate region
in mouse chromosome 17.
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Fig. 2.
Structure of the mTMT gene. The nucleotides that comprise the 5'-flanking region,
four introns, and 3'-UTR of the mTMT gene are in
lowercase letters, whereas those that comprise the five
translated exons of the mTMT gene are in uppercase
letters. Numbering of the nucleotides begins at the gene's
translation-initiation site because the 5'-UTR in exon 1 has not been
conclusively established. The exons are boxed and the
deduced amino acids of the initially translated product are indicated,
as well as the components of the catalytic triad ( ). The last
nucleotide in exon 6 of the mMCP-6 gene resides immediately
5' of the depicted sequence (i.e. residue  2340).
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Only one DNA fragment was detected when a mouse genomic DNA blot was
probed under conditions of high stringency with a probe derived from
exon 3 of the mTMT gene (Fig.
3A). Although this finding
indicates that the probe used in the DNA and RNA blot analyses is
relatively gene-specific, 3 to 5 genomic fragments were obtained when
another blot was probed under less stringent conditions (Fig. 3B).
Subsequent reprobing of these DNA blots indicated that the weaker
hybridizing fragments were not derived from the mMCP-6 or
mMCP-7 genes (data not shown). Thus, there appears to be at
least three mTMT-like genes in the mouse genome.
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Fig. 3.
Genomic blot analysis. Blots containing
mouse genomic DNA that had been digested with BamHI,
ScaI, HindIII, PstI, BglII,
SacI, AvrII, or XbaI were probed under
conditions of high (A) or moderate (B) stringency
with a radiolabeled 224-bp fragment derived from exon 3 of the
mTMT gene. This probe corresponds to amino acid residues 75 to 149 in the mature tryptase. DNA fragments of known molecular weight
(derived by Life Technologies, Inc. from a HindIII digest of
DNA) are indicated on the left of each blot.
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Isolation and Characterization of the mTMT Transcript and
Evaluation of Its Expression--
The steady-state level of the
mTMT transcript was below detection in BALB/c mouse-derived
V3 MCs (data not shown), as well as in BALB/c and 129/Sv mBMMCs (Fig.
4A). Nevertheless, the
corresponding mBMMCs developed from W/Wv and C57BL/6 mice
contained high levels of the mTMT transcript (Fig.
4A). Kinetic studies revealed that the mTMT
transcript is expressed quite early in the differentiation process of
uncommitted progenitors into immature MCs in the latter two mouse
strains. While these data indicated that mTMT is expressed
in mouse MCs in a strain-dependent manner, RNA blot
analysis of varied tissues of the C57BL/6 mouse also indicated that
mTMT is expressed in a tissue-dependent manner.
Of those analyzed tissues in the C57BL/6 mouse (Fig. 4B),
the intestine contained the highest level of mTMT mRNA.
The level of mTMT mRNA in the intestine of the BALB/c mouse is substantially lower than that in the intestine of the C57BL/6
mouse (Fig. 4B). Nevertheless, its presence indicates that
the mTMT gene can be transcribed in vivo in the
BALB/c mouse. The mTMT transcript was not abundant in leg
skeletal muscle which is rich in
mMCP-6+/mMCP-7+ MCs in the BALB/c mouse and
most other mouse strains.
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Fig. 4.
Kinetics of expression of the mTMT transcript in mBMMCs developed from four different mouse strains,
and expression of the mTMT in various tissues of two
adult mouse strains. A, interleukin
3-dependent mBMMCs were developed from BALB/c, C57BL/6,
129/Sv, and W/Wv mice. Total RNA was isolated weekly from
each culture. After 7 weeks of continuous culture, RNA blots were
prepared and analyzed with gene-specific probes for mTMT, mMCP-6,
mMCP-7, and -actin mRNA to evaluate the kinetics
of expression of the mTMT transcript in the mBMMCs developed
from each strain. The level of mMCP-7 mRNA is below
detection in C57BL/6 mBMMCs because of a point mutation in the exon
2/intron 2 splice site of the gene in this strain (38). B,
blots containing total RNA from C57BL/6 and BALB/c mouse ear, tongue,
spleen, kidney, lung, brain, skeletal muscle (leg), heart, intestine,
and liver were analyzed with gene-specific probes for mTMT
and mMCP-6 mRNA. The RNA gels used in these experiments
were also stained with ethidium bromide to demonstrate that comparable
amounts of 18 S rRNA were loaded into each lane.
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Although most of the mTMT transcripts in C57BL/6 mBMMCs and
intestine were ~1.2 kb in size, larger sized transcripts were occasionally detected. A search of the GenBank data base of ESTs was
therefore carried out in the initial attempt to obtain a full-length mTMT cDNA. An EST clone generated from mixed mouse tissue (GenBank accession number AA266560) was identified in the data base. Nucleotide
sequence analysis of this clone revealed that it possessed all but the
5' portion of the putative ~1.2-kb mTMT transcript depicted in Fig. 5. Using a RACE
approach, the remaining portion of the mTMT was isolated
from mouse liver. To confirm the nucleotide sequence of the authentic
mTMT transcript, a RT-PCR approach was then used to isolate a near
full-length clone from W/Wv mBMMCs. The cumulated data
(Fig. 5) revealed that the exon/intron boundaries of the 3.7-kb
mTMT gene (Fig. 2) were correctly predicted.
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Fig. 5.
Nucleotide and amino acid sequences of the
mTMT transcript. Three different molecular
approaches were used to deduce the nucleotide and amino acid sequences
of the 1.2-kb mTMT transcript. As noted under "Results,"
an EST was identified, isolated, and sequenced from mixed mouse tissues
that corresponds to 109 to 1112 of the mTMT transcript. A
5'-RACE approach was then carried out on a pool of liver cDNAs to
isolate a cDNA that corresponds to residues 7 to 626 in the
transcript. Finally a RT-PCR approach was used to isolate a cDNA
from mBMMCs that corresponds to residues 1 to 937 in the
mTMT transcript. The nucleotide and amino acid sequences of
the cDNAs are depicted. The two potential N-linked
glycosylation sites in mTMT are circled and its
COOH-terminal transmembrane segment is boxed. Components of
the catalytic triad (), translation-initiation site (*), signal
peptide (single bracket), and propeptide (double
bracket) are indicated. Nucleotide numbering begins at the first
nucleotide identified so far in the transcript; amino acid numbering
(within brackets at left) begins with residue 1 of the mature protein.
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Isolation and Characterization of the hTMT Gene and Transcript, and
Chromosomal Location of the hTMT Gene--
A search of the GenBank
data base of ESTs was carried out to determine whether or not there is
a human ortholog of the mTMT transcript. Two human EST
clones (GenBank accession numbers AA327025 and AA503882) were
identified in the data base which possessed short stretches of
nucleotides that were quite similar to those of the query mouse
sequence. Unfortunately, because neither clone was available from the
I.M.A.G.E. consortium, it was not possible to determine the nucleotide
sequences of their entire inserts. A PCR/RACE approach was therefore
employed to isolate the hTMT transcript. An oligonucleotide
that corresponds to a relatively conserved region in various MC
tryptase transcripts and an oligonucleotide that corresponds to the 3'
end of the mTMT transcript was initially used to isolate a
326-bp fragment of the hTMT transcript from human liver.
Based on the nucleotide sequence of this PCR product, more specific
oligonucleotides were synthesized and used in subsequent 5'- and
3'-RACE approaches to obtain the near full-length hTMT cDNA from liver, uterus, cecum, and skin (Fig.
6A). Analysis of the resulting
cDNAs revealed that the hTMT transcript has a
5'-untranslated region (UTR) which consists of at least 77 nucleotides
and a 3'-UTR that consists of >225 nucleotides. Like the
mTMT cDNA (Fig. 5), the hTMT cDNA (Fig.
6A) lacks a classical "AATAAA" polyadenylylation regulatory site in its 3'-UTR.
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Fig. 6.
Nucleotide and amino acid sequences of the
hTMT transcript, and evaluation of its expression at
the mRNA level in varied adult, fetal, and tumor tissues.
A, a PCR approach was used to obtain a near full-length
hTMT cDNA from human liver, cecum, uterus, and skin. The
consensus nucleotide and amino acid sequences of the PCR products are
depicted. The one potential N-linked glycosylation site in
hTMT is circled and its COOH-terminal transmembrane segment
is boxed. Components of the catalytic triad (),
translation-initiation site (*), signal peptide (single
bracket), and propeptide (double bracket) are
indicated. Nucleotide numbering begins in the 5'-UTR of the isolated
transcript; amino acid numbering (within brackets at
left) begins with residue 1 of the mature protein.
B, cDNA panels from CLONTECH were
used in a PCR approach to evaluate hTMT mRNA expression
in the indicated normal adult and fetal tissues. The indicated
transformed human cell lines from CLONTECH were
also evaluated for their expression of hTMT and
glyceraldehyde-3-phosphate dehydrogenase (G3PDH)
mRNA. These latter cell lines were maintained as solid tumors in
nude mice. The three indicated negative control PCRs () were carried
out in the absence of template DNA.
|
|
Analysis of varied human adult, fetal, and tumor tissues (Fig.
6B) suggests that the expression of hTMT at the
mRNA level is not as restricted as its mouse ortholog. Although the
hTMT transcript was found in many normal and tumor tissues,
it was not detected in skeletal muscle. The failure to detect the
hTMT transcript in adult spleen even though hTMT
mRNA is present in fetal spleen (Fig. 6B) raises the
possibility that the expression of this tryptase is developmentally
regulated in certain human tissues.
Based on the hTMT mRNA data, a PCR approach was used to
isolate and characterize the human gene (Fig.
7A). The exon/intron organizations of the hTMT and mTMT genes are
similar. Except for intron 1, the exons and introns of this gene are
comparable in size in the two species. Exon 1 encodes the hydrophobic
signal peptide predicted to be removed during the maturation of the
zymogen. Thus, as expected, exon 1 is the least conserved exon. Exons
2, 3, 4, and 5 of the hTMT and mTMT genes are 79, 79, 76, and 75% identical, respectively. Using the hamster/human
hybrid cell lines that vary in which human chromosome they contain, it
was discovered that the hTMT gene resides on chromosome 16 (Fig. 7B).
View larger version (50K):
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|
Fig. 7.
Structure and chromosomal location of the
hTMT gene. A, the nucleotides that
comprise the exons and introns of the hTMT gene are in
upper and lower-case letters, respectively. The
exons are boxed and the deduced amino acids of the initially
translated product are indicated, as well as the components of the
catalytic triad (). B, genomic DNA derived from 24 human/hamster somatic hybrid cell lines were used as templates in a PCR
approach to determine the chromosomal location of the hTMT
gene. Normal human and hamster genomic DNA were used as positive (+)
and negative () controls, respectively. As noted (arrow),
the relevant PCR product was only generated from the human/hamster cell
line that contained human chromosome 16. The 1-kb molecular weight
ladders are shown at both ends of the blot.
|
|
Amino Acid Sequence Analysis of mTMT and hTMT, and the Expression
of these Proteases in MCs--
mTMT is predicted to be translated as a
zymogen which consists of a signal peptide of 19 residues, a propeptide
of 10 residues, and a mature domain of 282 residues. The propeptides of
mTMT and hTMT do not resemble those in any other MC tryptase in terms
of their amino acid sequences (Fig. 8).
hTMT consists of 321 amino acids and has 10 more residues than mTMT
mainly due to an insertion of 9 amino acids in its prepropeptide. The
overall amino acid sequences of mature mTMT and hTMT are 74%
identical. When compared with other tryptases in the chromosome 17 family, mature mTMT is 45% identical to mMCP-6 and 46% identical to
mMCP-7; mature hTMT is 48% identical to human tryptase I. However, if
the dissimilar prepropeptide and COOH-terminal extension peptide of
mTMT and hTMT are taken into account, the extent of homology of this
new tryptase with the other members of its family is considerably lower. mTMT has two potential N-linked glycosylation sites
but these sites are not conserved in other mouse and human tryptases in
the family. At pH > 6.5, mature mTMT and hTMT have overall charges of 6 and 3, respectively. However, at pH < 6.5, mature mTMT and hTMT have overall charges of +4 and +5, respectively, and these positively charged residues are aligned predominately on one
face of each tryptase.
View larger version (83K):
[in this window]
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|
Fig. 8.
Comparison of the amino acid sequences of
mTMT and hTMT with each other and with other mouse and human MC
tryptases. The amino acid sequences of mMCP-6 (1, 2), mMCP-7 (3,
4), hTryptase-I (15), hTryptase-II/ (14, 15), hTryptase-III (15),
and hTryptase- (13) were extracted from SwissProt data base. The
depicted multiple sequence alignments were performed using the PILEUP
program of the Eugene "GCG" software package. In each instance,
identical amino acids in the sequences are shaded. Numbering
begins at the first residue in the mature portion of the tryptase. The
transmembrane segments of mTMT and hTMT are bracketed. The
seven putative loops (designated A-D and 1-3)
that form the substrate-binding pockets of these tryptases are
underlined.
|
|
Using an anti-peptide approach, hTMT-specific antibodies were generated
to confirm that the isolated transcripts are translated in certain
populations of human MCs. In control experiments, the antibodies
recognized recombinant hTMT zymogen but failed to recognize recombinant
mMCP-6, mMCP-7, mTMT, human tryptases , or human tryptase II/
(Fig. 9A). Since the
corresponding region in human tryptases I, II/, and III are 100%
identical, the antibodies also cannot recognize human tryptases I or
III. Using this highly specific antibody, immunoreactive hTMT was found
in the MCs that reside in human large intestine (Fig. 9B)
and skin (Fig. 9D). At the ultrastructural level, most of
the tryptase in the cutaneous MC resides in the secretory granules
(Fig. 9E).
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|
Fig. 9.
Immunohistochemistry. Replicate
SDS-PAGE/immunoblots containing similar amounts of the recombinant
tryptases mMCP-7, mMCP-6, human tryptase II/, human tryptase ,
mTMT, and hTMT were probed in A with anti-hTMT Ig (top
blot). Because each recombinant zymogen has the 8-residue FLAG
peptide attached to its COOH terminus, the anti-FLAG antibody was also
used in A to demonstrate that similar amounts of recombinant
tryptase is present in each lane (bottom blot). Even though
the overall amino acid sequences of hTMT and mTMT are 74% identical,
anti-hTMT Ig does not recognize mTMT or any other recombinant mouse or
human tryptase. Because anti-hTMT antibody is highly specific, human
intestine (B) and skin (D and E) were
stained with the antibody to evaluate the major cell types in these
tissues which contain hTMT protein. For a negative control, human
intestine was stained with an irrelevant anti-peptide rabbit antibody
(C). Human skin was stained with gold-labeled anti-hTMT
antibody to identify where hTMT resides in its expressing cell. As
noted in B and D, cutaneous and intestinal MCs
express hTMT protein (arrows) and most of this tryptase
resides predominately in the secretory granules (arrow). To
confirm that the immunoreactive cells in B are indeed MCs,
the adjacent serial section (data not shown) was stained with a
commercial antibody (Chemicon, Temecula, CA) that recognizes all human
tryptases but hTMT.
|
|
| |
DISCUSSION |
Complexes of tryptase genes reside on human chromosome 16 (16) and
the syntenic region of mouse chromosome 17 (17, 18). While analyzing
the mouse tryptase complex in greater detail, a novel 3.7-kb gene was
discovered that encodes an unusual transmembrane tryptase. No serine
protease has been discovered which possesses an overall structure that
resembles mTMT or hTMT.
Adjacent genes on a mammalian chromosome tend to be separated by ~30
kb of flanking DNA. Nevertheless, a cluster of five ~3-kb trypsinogen
genes that are separated by 7 kb or less of flanking DNA has been
identified in the human T cell receptor locus (30). Another cluster of
serine protease genes has been identified on mouse chromosome 14, some
of which are also separated by 5 to 7 kb of flanking DNA (17, 31, 39).
The observation that the chromosome 14 family of serine proteases has
an extremely low recombination frequency suggested that the close
spacing may be a common feature for the chromosomal organization of
serine protease genes within an individual family. The fact that more tryptases have been cloned from humans (13-16) than mice (1-4), coupled with the fact that the mMCP-6 and mMCP-7
genes are separated on chromosome 17 by ~1.2 centimorgans (17, 18),
raised the possibility that undiscovered tryptase genes might reside on
the chromosome between the mMCP-6 and mMCP-7
genes. Thus, a chromosome-walk approach was used to identify the
functional gene that is adjacent to the mMCP-6 gene. As
noted in Figs. 1 and 2, a novel tryptase gene was identified 2.3 kb 3'
of the mMCP-6 gene.
The isolation and characterization of the mTMT gene (Fig. 2)
and its transcript (Fig. 5) eventually led to the isolation and characterization of a related gene (Fig. 7) and transcript (Fig. 6) in
humans. Although genomic blot analysis revealed that the mouse genome
contains at least three mTMT-like genes (Fig. 3), the
isolated human gene (Fig. 7A) appears to be the ortholog of the mTMT gene because it possesses a high degree of sequence
identity throughout nearly all of its exons. Like the four other human MC-restricted tryptase genes (16), the hTMT gene resides on chromosome 16 (Fig. 7B).
While the level of the mTMT transcript was below detection
in BALB/c mBMMCs (Fig. 4A), the corresponding mBMMCs
developed from C57BL/6 and W/Wv mice contained high levels
of the transcript. Thus, mTMT mRNA is expressed in mice
in a strain-dependent manner. W/Wv mBMMCs also
differ from BALB/c mBMMCs in their expression of the chymases mMCP-2
and mMCP-4 (40). While it was initially thought that the
mMCP-2 and mMCP-4 genes were not transcribed in
BALB/c mBMMCs, subsequent studies revealed that these chymase transcripts are produced but often are rapidly degraded in this mouse
strain by a novel cytokine-dependent, post-transcriptional mechanism (41). It was then discovered that the expression of mMCP-7 mRNA in BALB/c mBMMCs is also regulated, in part,
by a cytokine-dependent post-transcriptional
mechanism.4 Repetitive motifs
residing in the 3'-UTR often regulate the stability of transcripts (42,
43). The finding that the 3'-UTR of the mTMT transcript has
cytosine-rich motifs (Fig. 5) which resemble those in mMCP-2
and mMCP-4 transcripts (41) raises the possibility that the
strain-dependent expression of mTMT mRNA is
also regulated, in part, by a post-transcriptional mechanism.
Of those analyzed tissues, the intestine contained the highest level of
the mTMT transcript (Fig. 4B). In most strains,
the MCs in the jejunal submucosa express mMCP-6 and
mMCP-7 (20). While the RNA data raised the possibility that
mTMT is coordinately expressed in vivo with one
or both tryptases, subsequent studies revealed that mTMT
expression in the BALB/c mouse is regulated in a
tissue-dependent manner independent of mMCP-6
and mMCP-7. For example, the level of mTMT
mRNA is below detection in skeletal muscle which contains large
numbers of mMCP-6+ MCs (Fig. 4B). While the
expression of the TMT transcript may be less restricted in
humans than in mice, it is of interest to note that hTMT
mRNA also could not be detected in skeletal muscle (Fig.
6B). The latter RNA data support the nucleotide and protein sequence data which suggested that the isolated human gene is the
ortholog of the mTMT gene.
mTMT and hTMT have all of the features of functional serine proteases
(Fig. 8) and immunohistochemical data (Fig. 9) confirmed that the human
transcripts are converted into protein. It is possible that hTMT is
expressed by other cell types. However, at least in the skin and large
intestine, its expression appears to be restricted to the MC. Both
putative tryptases have the His-Asp-Ser catalytic triad and the
NH2-terminal Ile that becomes buried in the activation
grove of a typical serine protease during its maturation (44, 45).
Loops 1 and 2 of the substrate-binding cleft are the most conserved
loops in the mouse tryptase family. Loop 1 is located at the base of
the S1 pocket and therefore forms a critical portion of the
substrate-binding cleft of serine proteases (44). Analogous to other
tryptases, the conserved Asp residue that dictates tryptic specificity
is present in loop 1 of mTMT and hTMT. These findings strongly suggest
that mTMT and hTMT are tryptases. When comparing the three cloned mouse
MC tryptases (Fig. 8), the other loops that form the substrate-binding
clefts of mTMT, mMCP-6, and mMCP-7 differ substantially in their amino acid sequences. In addition, loops A and C differ in their length. Thus, the preferred substrate preference of mTMT almost certainly differs from that of mMCP-6 and mMCP-7.
One of the most distinctive features of TMT is the transmembrane
segment located at its COOH terminus. Because this segment does not
have a Tyr residue in either mouse or human TMT, the COOH terminus
cannot undergo Tyr phosphorylation. However, because the cytosolic
domain has a conserved Ser residue, it is possible that this residue
undergoes phosphorylation during the metabolism of the tryptase. Other
tryptases have conserved Tyr-, Pro-, and Trp-rich domains which are
needed for tetramer formation (4, 45). Based on the crystallographic
structure of human tryptase II/, 6 surface loops are involved in the
two different kinds of contacts within the tetramer. Because these
loops are not conserved in sequence and length, it is unlikely that TMT
is able to form a similar tetramer structure. Nevertheless, this
putative tryptase has structural features that allow it to interact
with other proteins. Comparative structural modeling analysis revealed
that at pH < 6.5, mature mTMT and hTMT have a number of positive
charged residues that are aligned predominately on one face of the
tryptase. MC granule proteases use similarly positioned positively
charged faces to ionically bind to negatively charged serglycin
proteoglycans (25, 26, 46) and other granule constituents. Thus, hTMT and mTMT have the capacity of binding to certain negatively charged molecules during their biosynthesis and/or catabolism.
All mature human and mouse tryptases have 8 Cys residues that are
needed for the proper formation of 4 disulfide bonds in the folded
protease. These conserved Cys residues are found in mTMT and hTMT. mTMT
has an additional Cys residue, whereas hTMT has 4 additional Cys
residues. A tryptase has been cloned from dog mastocytoma (9) which has
4 more Cys residues than mMCP-6 and mMCP-7. The dog tryptase forms a
tetramer that consists of two 66-kDa dimers with each dimer having
disulfide-linked monomers (47). Thus, it is possible that the
additional Cys residues in TMT allows this tryptase to form dimers with
itself or another protein.
Those few membrane-associated serine proteases with a trypsin-like
specificity that have been found so far regulate diverse processes.
Whatever the function of TMT, the number of tryptase genes at the
chromosome 17 complex in mice, and the corresponding chromosome 16 complex in humans has been underestimated. A primordial serine protease
gene residing on chromosome 17 in mice (and the syntenic region of
chromosome 16 in humans) duplicated early and often during the
evolution of mammals to generate a panel of homologous tryptases in
each species that differ in their tissue expression, substrate
specificities, and physical properties.
| |
ACKNOWLEDGEMENTS |
We gratefully acknowledge the technical
assistance of Xuzhen Hu (Brigham and Women's Hospital, Boston, MA) and
the helpful suggestions of Dr. Nancy Kedersha (Brigham and Women's Hospital).
| |
FOOTNOTES |
*
This work was supported in part by Grants AI-23483,
GM-54762, HL-36110, and HL-63284 from the National Institutes of
Health, Grant BIR-9601845 from the National Science Foundation, and
Grants 970843 and 970949 from the National Health and Medical Research Council (Australia).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 and amino acid sequences reported in this paper
for the human TMT gene, human TMT cDNA, mouse TMT gene, and mouse
TMT cDNA have been submitted to the GenBankTM/EBI Data
Bank with accession numbers AF175759, AF175522, AF175760, and AF175523,
respectively.
¶
Sinsheimer Scholar (Alexandrine and Alexander L. Sinsheimer
Fund) and an Alfred P. Sloan Research Fellow.
To whom correspondence 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.
2
R. L. Stevens, unpublished observations.
3
The three-dimensional models for mTMT and hTMT
can be downloaded from the Web at
http://guitar.rockefeller.edu/pub/sali/models.
4
R. L. Stevens, unpublished data.
| |
ABBREVIATIONS |
The abbreviations used are:
MC, mast cell;
bp, base pair;
EST, expressed sequence tag;
Ig, immunoglobulin;
mBMMC, mouse bone marrow-derived MC;
mMCP, mouse MC protease;
PCR, polymerase
chain reaction;
RACE, rapid amplification of cDNA ends;
RT, reverse
transcription;
TMT, transmembrane tryptase;
UTR, untranslated region;
kb, kilobase..
| |
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