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-Tryptases, Novel Members of the Chromosome 16p Mast Cell Tryptase and Prostasin Gene Families1
Cardiovascular Research Institute and Department of Medicine, University of California, San Francisco, CA 94143
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResults and DiscussionReferences |
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-, ß-, and
mast cell protease-7-like tryptase genes on human chromosome 16p13.3.
The present work characterizes adjacent genes encoding novel serine
proteases, termed 
-tryptases, and generates a refined map of the
multitryptase locus. Each gene lies between an 1H
Ca2+ channel gene (CACNA1H) and a ßII- or
ßIII-tryptase gene and is 
30 kb from polymorphic minisatellite
MS205. The tryptase locus also contains at least four tryptase-like
pseudogenes, including mastin, a gene expressed in dogs but not in
humans. Genomic DNA blotting results suggest that I- and
II-tryptases are alleles at the same site. ßII- and
ßIII-tryptases appear to be alleles at a neighboring site, and II-
and ßI-tryptases appear to be alleles at a third site. -Tryptases
are transcribed in lung, intestine, and in several other tissues and in
a mast cell line (HMC-1) that also expresses -tryptase protein.
Immunohistochemical analysis suggests that -tryptase is expressed by
airway mast cells. -Tryptase catalytic domains are 48% identical
with those of known mast cell tryptases and possess mouse homologues.
We predict that -tryptases are glycosylated oligomers with tryptic
substrate specificity and a distinct mode of activation. A feature not
found in described tryptases is a C-terminal hydrophobic domain, which
may be a membrane anchor. Although the catalytic domains contain
tryptase-like features, the hydrophobic segment and intron-exon
organization are more closely related to another recently
described protease, prostasin. In summary, this work describes
-tryptases, which are novel members of chromosome 16p
tryptase/prostasin gene families. Their unique features suggest
possibly novel functions. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Several studies suggest roles for tryptases in allergic airway disease. Lavage tryptase levels rise in asthmatics following endobronchial allergen challenge (8). In dog and human bronchi, tryptases increase histamine-induced bronchoconstriction (9, 10) and also may promote constriction by degrading vasoactive intestinal peptide (4). By stimulating airway smooth muscle cell (11) and fibroblast growth (12, 13), as well as chemotaxis and collagen synthesis (14, 15), they may contribute to muscle hypertrophy and subepithelial fibrosis in asthma. The hypothesis that tryptases worsen asthma is buttressed by reports that tryptase inhibitors block allergic bronchoconstriction and eosinophilic inflammation in sheep (16) and, in the first human trials of tryptase inhibitors, reduce asthmatic responses to inhaled allergen in asthmatics (17).
Known human tryptases divide into two groups, and ß. -Tryptase
is the major circulating isoform and may be the main type expressed by
basophils (18, 19). ß-Tryptases appear to be the major
type stored in secretory granules and are the major form isolated from
lung extracts and mast cells purified from lung and skin
(19). Four different human cDNAs (, ßI, ßII, and
ßIII) have been isolated from lung and skin mRNA
(20, 21, 22). ß-Tryptases are 98–99% identical in amino
acid sequence. -Tryptases are less closely related (I is 91%
identical with ßI). Our laboratory reported recently that II,
ßI, ßII, and ßIII genes are clustered on chromosome 16p13.3 along
with genes encoding novel tryptases, the fifth exon of which is related
to the mouse tryptase, mouse mast cell protease
(mMCP)-73
(23). The intron-exon organization of tryptase genes is
unique, suggesting that tryptases are a distinct and perhaps ancient
branch of the trypsin clan of serine peptidases.
In mice, tryptases mMCP-6 and -7 have been identified (24, 25). Structurally, human /ß-tryptases are more closely
related to each other than to mMCP-6 and -7, which are much more
different from each other than any combination of known human
tryptases. This suggests that mMCP-6 and -7 are not the equivalents,
respectively, of - and ß-tryptases and that ancestors of
/ß-tryptases diverged from each other after the point when known
mouse and human tryptases shared a common ancestor (23, 26). In dogs, our laboratory characterized a mast cell protease
termed mastin, a relative of tryptase sufficiently different from known
tryptases that it forms a separate branch of the tree
(27, 28, 29). No expressed human or mouse homologue has been
identified.
Here we demonstrate expression of novel human tryptases, termed
because they are distinct from , ß, and other human tryptases. We
also present a refined map of /ß genes and identify a gene
encoding a human homologue of dog mastin.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Human ßI-tryptase cDNA (22) and gene sequence (23) and Basic Local Alignment Search Tool (www.ncbi.nlm.nih.gov) algorithms were used to query expressed sequence tag (EST) and high throughput genome sequence (HTGS) in GenBank. Predicted novel human cDNAs were used to query GenBank’s mouse EST database to identify mouse homologues.
Cell culture
Cells from the human mast cell line HMC-1 (5C6 subclone, kindly
provided by Dr. Beate M. Henz) were cultured as described
(30). HMC-1 cells express active ßI-tryptase but not
-tryptase (19, 30, 31, 32).
Amplification and cloning of cDNAs
cDNA predicted from EST and genomic sequence was used to design
PCR primer pairs, which then were used to screen cDNAs prepared from a
spectrum of human tissues for expression of -tryptases. Sequencing
of amplimers allowed us to prove the identity of the PCR-derived bands,
to confirm exon sequences predicted from genomic and EST DNA, and to
confirm intron-exon splice site predictions. Amplified fragments of
-tryptase cDNA from human lung and from HMC-1 5C6 cells were
subcloned into pCR2.1 (Invitrogen, Carlsbad, CA) for further sequencing
and for generation of specific probes to use in blotting studies. Bands
containing amplimers from colon, small intestine and testis were
sequenced directly.
DNA and protein sequence comparisons
DNA sequencing was conducted by University of California at San Francisco’s Biomolecular Resource Center using standard dideoxy techniques with fluorescent dye terminators. Multiple sequence alignments, matrix analyses, and dendrograms were generated using GeneWorks software (Oxford Molecular, Campbell, CA).
Chromosomal localization and screening of bacterial artificial chromosomes (BACs)
BAC clones 324 and 48 previously were identified and localized
to chromosome 16p13.3 as described (23). In the current
study, -tryptase genes were detected in these BACs by PCR of
gene-specific fragments and by DNA blotting using the general
approaches we described previously in connection with these BACs
(23). BAC fragments generated by digestion with
HindIII were subcloned. Genes were mapped to specific sites
in BAC 
subclones by blotting of electrophoresed DNA restriction
digests and by sequencing of PCR amplimers, BAC ends, and portions of
selected subclones.
mRNA and DNA blotting
mRNA extracted and purified from cultured HMC-1 5C6 cells was
electrophoresed in agarose and blotted to nylon membranes. These blots,
along with blots of mRNA from a range of human tissues (Invitrogen),
were hybridized with radiolabeled cDNA probes corresponding to
protein-coding exons. BAC and full human genomic DNA were digested with
restriction endonucleases, size-fractionated in agarose, then blotted,
baked, and prehybridized as described (23). The resulting
blots were probed with a radiolabeled 1100-bp fragment of the BAC 48
-tryptase gene bracketing the internal NotI site, or with
labeled subclones of this fragment lying on each side of the
NotI site, then subjected to autoradiography.
Ab generation, immunoblotting, and immunohistochemistry
Polyclonal Abs recognizing human -tryptase were raised in
chickens immunized with a keyhole limpet hemocyanin-conjugated
synthetic peptide (CRRDYPGPGGSILQP) corresponding to residues 192–206
of human prepro--tryptase. Conjugations and immunizations were
conducted by AnaSpec (San Jose, CA). In preparation for immunoblotting,
HMC-1 5C6 cell proteins were extracted into detergent solution (4% SDS
in 0.2 M DTT, 20% glycerol, 0.125 M Tris, pH 6.8). Extracts were
electrophoresed in 12.5% SDS-polyacrylamide gels and electroblotted to
polyvinylidene difluoride membranes, which were preincubated with 0.3%
Tween-20 in 50 mM Tris (pH 7.2) and 0.5 M NaCl, then hybridized with
1:1000 dilutions of chicken antiserum for 1 h in the same buffer.
Bound Abs were detected with goat anti-chicken IgG-alkaline
phosphatase (1:5000) and Fast Red TR/Naphthol AS-MX Phosphate (Sigma,
St. Louis, MO). Control blots were incubated with preimmune chicken
serum or with secondary Ab.
For immunohistochemical analysis using the same anti--tryptase
antiserum, samples of large airways were obtained (with approval of the
University of California at San Francisco Committee on Human Research)
from patients undergoing lung transplantation for cystic fibrosis.
Five-micrometer airway cryosections placed onto glass slides were
equilibrated in 0.3% H2O2
and 90% methanol for 10 min, washed with PBS, then incubated for 15
min with blocking solution (PBS containing 5% dehydrated milk, 3%
nonimmune goat serum, 0.1% Triton X-100, and 1% glycine) at 18°C.
Blocking solution was removed and tissues probed either with a 1:100
dilution of chicken nonimmune serum or anti--tryptase antiserum
overnight at 4°C. Tissues were then washed in PBS plus 0.05%
Tween-20, incubated with a 1:200 dilution of alkaline
phosphatase-conjugated goat anti-chicken IgY Ab (Promega, Madison,
WI) for 10 min at 18°C, then washed again. Bound alkaline phosphatase
was detected using the substrate 5-bromo-4-chloro-3-indolyl
phosphate/nitroblue tetrazolium (Zymed Laboratories, South San
Francisco, CA).
Molecular modeling
We constructed a homology-model of the I-tryptase catalytic
domain with assistance of an automated protein modeling tool and server
(Swiss PDB Viewer and Swiss-Model, respectively) (33). The
pro- and C-terminal hydrophobic sequences were excluded from the model.
X-ray diffraction-derived coordinates of human ßII-tryptase (1AOL)
(34) served as template for the model, which was optimized
by idealizing bond geometry and removing unfavorable nonbonded
contacts.
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Results and Discussion |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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-tryptase cDNAs and prediction of amino acid
sequence
cDNAs encoding parts of human -tryptase were obtained initially
by screening EST databases using known tryptases as query sequences.
Because these sequences are partial and disagree in certain areas of
overlap, PCR primers based on EST sequence were used to amplify more
complete cDNAs from human lung cDNA. The full amino acid sequence of
321 amino acids deduced from a combination of PCR-derived lung cDNA and
EST sequence is identical with that predicted for gene-derived
I-tryptase in Fig. 1
, which compares
gene-derived prepro--tryptase primary sequences with those of
related serine proteases. The I and II structures aligned in Fig. 1 represent amino acid sequences predicted from separate BAC genes (see
below). The predicted m.w. of prepro-I-tryptase is 33,689 without
glycosylation or other posttranslational modification. Based on
database searches and computer-assisted multiple sequence alignments
and phylogenetic comparisons, the I-tryptase catalytic domain amino
acid sequence is most similar to that of known mast cell tryptases,
exhibiting 47% identity to ßII-tryptase. Some structural features,
e.g., LPPPY (residues 173–177), are particularly tryptase-like. Other
features are common to all active serine proteases with tryptic
specificity, such as "catalytic triad" residues
His78 (His57, by standard
chymotrypsinogen numbering), Asp125
(Asp102, standard numbering), and
Ser222 (Ser195, standard
numbering), and also residue Asp216
(Asp189, standard numbering), which is the prime
determinant of specificity for Arg or Lys on the N-terminal side of the
scissile bond of peptide substrates. These features, along with the
clustering of -tryptase genes with known tryptase genes and evidence
of mast cell expression, are the basis of labeling these enzymes
tryptases. However, the catalytic domains are related almost as closely
to the recently described proteases prostasin (35) and
testisin (36). Indeed, when full preprosequence and gene
structures (see below) are compared, -tryptase appears to be more
similar to prostasin and testisin than to /ß-tryptases. Features
shared with prostasin and testisin include a propeptide ending in Arg
and a predicted C-terminal membrane anchor, neither of which are
present in /ß-tryptases, and a gene organization more
similar to that of prostasin than of previously described tryptases
(see below).
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-tryptase is activated directly from its catalytically inactive
zymogen form by a tryptic protease, which will sever the propeptide
from the catalytic domain, allowing it to adopt an enzymatically active
conformation. This mode of activation would differ from that of human
ß-tryptases, which lack a terminal propeptide basic residue and are
activated in a two-step process involving tryptase and dipeptidyl
peptidase I (37). Mature, active -tryptase, unlike
known tryptases, may be a two-chain protein, with part of the
propeptide remaining covalently attached via a disulfide linkage
involving Cys26 (Cys1 in
chymotrypsinogen) by analogy to the propeptide-catalytic domain linkage
in activated chymotrypsin (38). Prostasin and testisin
also contain this predicted linkage. It is possible that the
-tryptase propeptide will be removed through the action of
exopeptidases such as dipeptidyl peptidase I, which is highly expressed
in mast cell secretory granules (39). Based on a consensus
site at Asn85, mature -tryptases may be
N-glycosylated. The mature protein is likely to be an active protease
with tryptic specificity, based on conservation of critical triad
residues and of specificity-determining Asp216
(Asp189, by chymotrypsinogen numbering), as noted
above. Due to conservation of the LPPPY sequence found to form
noncovalent contacts between subunits in the ßII-tryptase tetramer
(34), -tryptase may form oligomers of catalytically
active units. However, -tryptase lacks the pair of Tyr residues that
also are predicted to promote LPPPY-mediated oligomerization.
Therefore, it is not yet possible to predict with certainty whether
-tryptase will oligomerize, like all heretofore-characterized mast
cell tryptases and close relatives, such as mastin
(29).
-Tryptases I and II contain a C-terminal extension with a highly
hydrophobic segment long enough to form a single-pass transmembrane
helix, as shown by the hydropathy plot in Fig. 2. This predicts that the catalytic
domain will be C-terminally membrane anchored. Within the endoplasmic
reticulum, Golgi, and secretory granules, the -tryptase catalytic
domain should lie in the lumen. If -tryptase traffics to the cell
surface, then the catalytic domain will lie on the extracellular
surface. The hydropathy analysis predicts a small cytoplasmic tail of
uncertain significance. The 10 residues on the N-terminal side of the
hydrophobic tail embody features typical of GPI-anchored proteins
(40). Possibly, the transmembrane sequence in mature
-tryptase is severed in the endoplasmic reticulum and replaced with
a GPI membrane anchor. If this is the case, -tryptase could be
released in a soluble form by GPI-specific phospholipases. The
predicted m.w. of the unglycosylated I-tryptase catalytic domain
plus C-terminal extension is 30,063. Without the C-terminal extension,
the predicted m.w. is 25,000. The deduced amino acid sequences of
-tryptases I and II are 98–99% identical with that of recently
described "transmembrane tryptase" (41).
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-tryptase cDNA, we identified mouse ESTs (e.g.,
AI466869 and AA266560) collectively encoding 158 residues of contiguous
sequence in the catalytic domain of a related mouse enzyme. Based on an
amino acid identity of 74% compared with human -tryptase in
regions of overlap, the mouse products are likely to be orthologs of
human -tryptases. Whether they are products of more than one gene
remains to be determined. This level of identity is similar to that
between human -tryptases and the mouse tryptases mMCP-6 and -7
(23). Among features conserved between mouse and human
enzymes are catalytic triad residues (His and Asp but not Ser are
covered by the partial mouse sequence), the consensus N-glycosylation
site, and five of six cysteines.
Tissue expression of -tryptase mRNA
As shown by the results of PCR in Fig. 3, we identify transcription of human
-tryptase in multiple tissues, including lung and small intestine.
However, several tissues screened using the same conditions yield no
signal or faint signals, suggesting tissue-specific differences in
levels of -tryptase transcription. Tissues expressing no detectable
-tryptase mRNA include peripheral blood leukocytes and thymus, a
finding that suggests that -tryptases are not highly expressed in
circulating immune cells. In contrast, -tryptase mRNA is abundant in
the 5C6 subclone of HMC-1 cells, which also express active
ßI-tryptase (19, 30, 31, 32). Sequencing of PCR-generated
amplimers from HMC-1 cells reveals the same sequence predicted from
amplimers of human lung cDNA. These sequences, which cover 70% of the
protein sequence predicted from BAC genes, is 99.1–97.8% identical
with that of I- and II-tryptase, respectively (see Fig. 1
) in 224 residues of overlap. Blotting of electrophoresed human mRNA
from a number of tissues does not yield strong hybridization signals
(not shown) with -tryptase probes, suggesting that overall levels of
-tryptase transcript are low (although detectable by PCR), perhaps
because -tryptase is expressed in a small subpopulation of cells in
the tissues surveyed. However, -tryptase mRNA is detected by
standard blotting of mRNA from the 5C6 subclone of HMC-1 cells (not
shown). The identification of -tryptase mRNA in a mast cell line
suggests that mast cells may be among the subset of cells expressing
-tryptases.
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-tryptase genes
Previously, this laboratory identified two BAC clones containing
clustered /ß-tryptase genes localizing to chromosome 16p13.3
(23). Overlapping portions of these BACs are homologous
but not identical. Hypothesizing that additional tryptase-like genes
lie in these two BACs (324 and 48 in Fig. 4), we screened both BACs for the
presence of -tryptase genes via PCR using EST- and cDNA-based
primers. Each BAC yields a strong band of amplimers, one of which was
subcloned and sequenced, confirming the presence of genomic sequence
containing the exon sequence predicted by -tryptase ESTs. To
localize -tryptase genes within each BAC, radiolabeled probes based
on cloned fragments of -tryptase were hybridized with BAC DNA
digested singly and in combination with HindIII,
NotI, and EcoRI. In each BAC clone, the results
localized a -tryptase gene to a particular HindIII
fragment bracketing a NotI site splitting the gene, as shown
in Fig. 4. The NotI site in the two -tryptase genes was
localized by sequencing genomic fragments generated by PCR. The
transcriptional orientation of each -tryptase gene was determined by
DNA blotting using radiolabeled probes derived from the 5' side of the
established NotI site. The I-tryptase gene on BAC 324
lies in a region covered by a subcloned HindIII fragment
(324D) found previously to contain the ßII gene (23).
On BAC 48, the nearest tryptase neighbor is the ßIII gene, which
resides on an adjacent HindIII fragment (48 M) rather
than on the same HindIII fragment due to the presence of a
HindIII site not present in the otherwise homologous region
of BAC 324. As shown in Fig. 5, DNA blots
of HindIII-digested BAC 324, BAC 48, and full genomic DNA
reveal that the HindIII isoforms predicted by the two BACs
both are present in some but not all individuals, suggesting that I
and II are alleles of each other. By inference, based on the
homologies between BAC 324 and 48 depicted in Fig. 4, other allelic
pairs of tryptases are ßII/ßIII, II/ßI, and mMCP-7-like I/II,
respectively.
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-tryptase ESTs and sequenced portions of the
BAC-derived genes, the nearest neighboring gene on the 3' flank is an
1H T-type Ca2+ channel gene identical or
closely related to CACNA1H. In the case of the BAC 324
I-tryptase gene, the 3' untranslated region possibly overlaps with
that of CACNA1H, which is in the opposite transcriptional
orientation, as depicted in Fig. 4. CACNA1H has been
localized to chromosome 16p13.3 by fluorescent in situ hybridization
(42). A mouse homologue (Cacna1h) has been
mapped to chromosome 17 in a region that is homologous with a portion
of human chromosome 16p13.3 (42). This syntenic region
also harbors two mouse tryptase genes, Mcpt6 and
Mcpt7 (43). At least two genomic fragments
encoding parts of CACNA1H were identified in database
searches. One of these (GenBank AL031703.9) aligns with one end of BAC
324 based on shared restriction patterns and a segment of near-identity
with BAC 324’s sequenced T7 end, as shown in Fig. 4, allowing
extension of the I-tryptase locus beyond the BAC terminus. A second
GenBank sequence (fragment 10 of unfinished chromosome 16-derived
contig AL031715.1) contains additional CACNA1H -related
exons and aligns elsewhere on BAC 324, as shown in Fig. 4. The 5' end
of CACNA1H predicted by cDNA cloning was not found in the
AL031703.9 sequence; therefore, the extended BAC 324 sequence in Fig. 4
does not include the entire gene. However, AL031703.9 contains the
well-known minisatellite MS205 (also known as DS16S309), which is
highly polymorphic and localizes to 16p13.3, 1.2 mb from 16pter
(44). MS205, which has been used to explore the origin of
human populations (45), is 30 kb from the
I-tryptase gene.
BAC 48, which contains the II-tryptase gene, also contains an 1H
T-type Ca2+ channel gene, based on sequencing of
the II gene itself (see below) and of the BAC insert’s SP6 end (see
Fig. 4), which contains part of a late CACNA1H-like exon in
the sequenced portion. This finding strengthens the finding of homology
between the two BACs based on restriction mapping and alignment of
tryptase genes. BAC 48’s T7 end extends beyond the area of homology
with BAC 324. The sequence of the T7 end is almost identical with that
of fragment 5 of a partially sequenced contig (GenBank AL031711.16),
which encodes three tryptase-like genes, MP-2, MP-7, and mastin, as
shown in Fig. 4. All three genes have multiple flaws, e.g., premature
stop codons, faulty intron-exon junctions (not shown). None have
closely related ESTs. Thus, they are likely to be pseudogenes. The
mastin gene, whose exons are most closely related to the tryptase-like
gene mastin characterized in dogs, is discussed in more detail
below.
-Tryptase gene sequence and organization
The BAC 324 and 48 -tryptase genes, termed I- and
II-tryptase, respectively, were fully sequenced to reveal their
relationship to the cDNAs in EST databases and in DNA amplified by PCR.
The GenBank accession numbers of the I and II genes are AF191031
and AF195508, respectively. In these genes, intron-exon splice
junctions were identified using open reading frames and cDNA alignments
by application of the "GT... AG" rule for initiating and ending
introns and by referring to patterns of intron phase and placement in
known tryptases and in other serine protease genes, as described
(23). As revealed in Fig. 5, we find that fragment 12 of
the unfinished chromosome 16-derived contig AL031715.1 deposited in the
HTGS database contains the partial sequence (exons 2–6) of a gene that
is homologous to the I- and II-tryptase genes described here.
However, two of the exons contain premature stop codons. It remains to
be established whether this gene is an allele of I and II or is a
gene at a separate site; in either case, assuming that this unfinished
sequence is accurate, it most likely corresponds to a pseudogene, given
the early stop codons. The simplest explanation of our data generated
from BACs 324 and 48 and from blotting of full genomic DNA is that
there is one genomic site with multiple alleles. As shown in Fig. 6 and discussed in the accompanying
legend, the organization of I/II introns and exons more closely
resembles that of the human prostasin gene (46) than of
/ß-tryptases, which are more compact genes featuring only five
protein-coding exons, with exon 1 consisting entirely of the 5'
untranslated region. Prostasin and the -tryptase genes each
distributes DNA encoding the prepropeptide over the first three exons,
with conserved intron size, phase, and placement. Additionally,
prostasin and -tryptase genes each encodes a C-terminal, putative
membrane-anchoring segment not found in any of the known
/ß-tryptases. Even though the nearest neighbors of -tryptase
genes are /ß-tryptases and they share certain tryptase-like
features, it is possible that -tryptases are phylogenetically more
closely related to prostasin than to /ß-tryptases. It is also
possible that ancestors of both prostasin and /ß-tryptases have
contributed to the present -tryptase genes through recombination,
exon swapping, and gene conversion mechanisms, such as those suggested
to lead to generation of human mMCP-7-like tryptases, which appear to
be chimeras of /ß-tryptase genes and an ancestral mMCP-7-like gene
(23).
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The sequence of a putative human homologue (not shown) of dog
mastin was predicted from the GenBank-derived genomic sequence. Intron
splice junctions were predicted by reference to dog mastin cDNA
(27) and patterns of intron phase and placement in known
tryptase genes (22, 23) by examination of open reading
frames and by application of "GT... AG" rules for intron ends,
as noted above in connection with -tryptase genes. As shown in Fig. 4, genomic sequences more homologous to dog mastin than to any other
known cDNA were identified in fragment 5 of partially sequenced contig
AL031711.16, which overlaps extensively with BAC 48’s T7 end. However,
no closely related human ESTs were found. The gene itself is flawed by
faulty splice junctions, by mutation of Ser195 to
Asn (which would lead to catalytically inactive protein if the gene
were expressed), and intronic insertions of Alu repetitive sequences.
Thus, this mastin-homologous gene is a pseudogene, which may explain
the absence of human ESTs and the failure of polyclonal antisera raised
against dog mastin (28) to identify homologues in human
tissues and cell lines (Ref. 29 and our unpublished
results). Two additional apparent tryptase pseudogenes (MP-2 and MP-7;
see Fig. 4) lie in the same fragment of AL031711.16. BAC 48’s T7 end
terminates at a HindIII site in a minisatellite in the
middle of MP-2 pseudogene intronic sequence. Thus, both BACs and their
flanks are rich in tryptase-like genes and pseudogenes. Based on the
sequence acquired so far, these regions of chromosome 16 are also
richly endowed with various repeats, at least one of which, MS205, is
highly polymorphic. DNA instability in the neighborhood of these
repeats may contribute to general instability in the region,
facilitating duplications, gene conversion events (23),
and proliferation of pseudogenes.
-Tryptase protein expression
Polyclonal Abs raised against a portion of the -tryptase
catalytic domain sequence recognize a protein of 31 kDa in extracts
of HMC-1 5C6 cells, as shown in Fig. 7.
This band is distinct from those of ß-tryptases based on its smaller
apparent size, narrower banding pattern, and lack of reactivity with
antisera raised against purified human lung mast cell tryptase (not
shown). These results suggest that one or more -tryptases are
expressed in 5C6 cells and predict that mast cells are a source of this
enzyme. The detection of -tryptase mRNA in a variety of tissues by
PCR, but not by conventional mRNA blotting, is consistent with the
expression of -tryptase in a subpopulation of cells found in many
tissues, such as mast cells. No difference in migration of the
immunoreactive band was found in the presence or absence of a reducing
agent, suggesting the absence of disulfide-linked oligomerization
characteristic of some tryptase-like enzymes, such as mastin
(29). The lack of a reduction in size in the presence of
reducing agents suggests that the predicted disulfide-linked propeptide
remnant is absent in the mature enzyme or that it is too small to
produce detectable differences in electrophoretic migration between
reduced and unreduced samples.
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, our polyclonal
anti--tryptase Abs recognize scattered, granulated mononuclear
cells in human airway submucosa. The distribution and appearance of
these cells is typical of tissue mast cells, which is consistent with
the identification of immunoreactive -tryptase in the 5C6 subclone
of the HMC-1 line of mast cells. The granular pattern of staining
suggests the possibility that immunoreactive -tryptase resides in
secretory granules.
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-Tryptase homology model
Models of the I-tryptase catalytic domain based on the
established structure of its closest crystallized relative,
ßII-tryptase, are depicted in Fig. 9.
These models reveal that the segment
Cys192-Pro206, which is
among the most distinctive compared with otherwise similar relatives
(see Fig. 1), forms a predicted surface loop that is far enough removed
from the classic serine protease substrate binding site that it is
unlikely to interact directly with polypeptide targets in the vicinity
of the scissile bond. Because this is the peptide segment against which
antisera were raised, it is unlikely that our anti-I-tryptase Ig
will block access to potential peptide substrates. The models also show
that the sole predicted N-glycosylation site is closer to the catalytic
active site. However, this glycosylation site, which is not conserved
in /ß-tryptases, prostasin, or testisin, lies outside of the
classic serine protease-extended peptide binding site. Carbohydrate
attached at this site could interfere with bulky protein substrates but
probably not with smaller peptide substrates. The models also predict
that the two cysteines (145 and 154) that we are unable to pair with
other cysteines based on the ßII-tryptase crystal structure are not
close enough to form a disulfide linkage with each other. Both lie on
predicted surface loops and could potentially form intersubunit bonds
with other catalytic subunits. However, there is no evidence of this in
HMC-1 immunoblots. As noted above, we predict that
Cys145 (Cys122 in
chymotrypsinogen) may remain linked to cleaved remnants of propeptide
by analogy to chymotrypsin. The model also predicts that the putative
membrane-anchored C-terminal hydrophobic segment attaches to the
catalytic domain’s back side, where it is less likely to interfere
with substrate access to the active site.
|
-tryptases to transmembrane tryptase
While this manuscript was under review, Stevens and colleagues
published a report of a similar gene, termed transmembrane tryptase
(47). Their gene contains one fewer exon and is 0.8 kb
shorter than our genes. However, the deduced amino acid sequence of
transmembrane and -tryptase catalytic domains is 98–99% identical,
suggesting that the enzymes may be allelic variants of each other.
Partial characterization by Stevens and colleagues of a gene locus
containing a mouse homologue closely related to that predicted from our
murine ESTs suggests that the mouse gene has a relationship to the
neighboring mouse tryptase mMCP-6 gene that is similar to the
relationship between the human II and ßIII tryptase genes we
describe here. Interestingly, Abs raised against transmembrane tryptase
recognize mast cells in human skin and intestine, thereby supporting
our hypothesis that -tryptase is expressed in mast cells based on
our identification of immunoreactive protein in human airway cells and
in a mast cell line. If, like other human tryptases, -tryptases are
expressed in most mast cells, then -tryptase expression in tissues
will be widespread. The immunological significance of -tryptase
expression by mast cells is not yet clear and awaits further
characterization of this novel gene product’s biogenesis and physical
and enzymological properties. If -tryptases, like mast cell
ß-tryptases, are activated intracellularly, stored in secretory
granules, and secreted in response to Ag-bound IgE, they may aggravate
the local pathology of allergic inflammation by hydrolyzing proteins in
the vicinity of degranulated mast cells. In this regard, if
-tryptases remain membrane-anchored after activation and release,
they may remain attached to the cell surface, thus ensuring that
proteolytic effects remain local. However, if -tryptases are
released from their membrane anchors, secretion may allow the enzymes
to reach targets that are remote from the site of mast cell
stimulation.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. George H. Caughey, Cardiovascular Research Institute, University of California at San Francisco, Box 0911, San Francisco, CA 94143-0911.
3 Abbreviations used in this paper: mMCP, mouse mast cell protease; EST, expressed sequence tag; HTGS, high throughput genome sequence; BAC, bacterial artificial chromosome.
Received for publication October 20, 1999. Accepted for publication March 28, 2000.
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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form of human tryptase is the predominant type present in blood at baseline in normal subjects and is elevated in those with systemic mastocytosis. J. Clin. Invest. 96:2702.
RI, and FcRI mRNAs in human mast cells and basophils by competitive reverse transcription-polymerase chain reaction. J. Immunol. 154:5472.[Abstract]
-chymotrypsin. V. The atomic structure of tosyl--chymotrypsin at 2 Å resolution. J. Mol. Biol. 68:187.[Medline]
1H from human heart, a member of the T-type Ca2+ channel gene family. Circ. Res. 83:103.This article has been cited by other articles:
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M. C. M. Strik, A. Wolbink, D. Wouters, B. A. Bladergroen, A. R. Verlaan, I. S. van Houdt, S. Hijlkema, C. E. Hack, and J. A. Kummer Intracellular serpin SERPINB6 (PI6) is abundantly expressed by human mast cells and forms complexes with {beta}-tryptase monomers Blood, April 1, 2004; 103(7): 2710 - 2717. [Abstract] [Full Text] [PDF]
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H.-W. Wang, H. P. McNeil, A. Husain, K. Liu, N. Tedla, P. S. Thomas, M. Raftery, G. C. King, Z. Y. Cai, and J. E. Hunt {delta} Tryptase Is Expressed in Multiple Human Tissues, and a Recombinant Form Has Proteolytic Activity J. Immunol., November 1, 2002; 169(9): 5145 - 5152. [Abstract] [Full Text] [PDF]
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G. W. Wong, P. S. Foster, S. Yasuda, J. C. Qi, S. Mahalingam, E. A. Mellor, G. Katsoulotos, L. Li, J. A. Boyce, S. A. Krilis, et al. Biochemical and Functional Characterization of Human Transmembrane Tryptase (TMT)/Tryptase gamma . TMT IS AN EXOCYTOSED MAST CELL PROTEASE THAT INDUCES AIRWAY HYPERRESPONSIVENESS IN VIVO VIA AN INTERLEUKIN-13/INTERLEUKIN-4 RECEPTOR alpha /SIGNAL TRANSDUCER AND ACTIVATOR OF TRANSCRIPTION (STAT) 6-DEPENDENT PATHWAY J. Biol. Chem., October 25, 2002; 277(44): 41906 - 41915. [Abstract] [Full Text] [PDF]
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J. K. Brown, C. A. Jones, L. A. Rooney, G. H. Caughey, and I. P. Hall Tryptase's potent mitogenic effects in human airway smooth muscle cells are via nonproteolytic actions Am J Physiol Lung Cell Mol Physiol, February 1, 2002; 282(2): L197 - L206. [Abstract] [Full Text] [PDF]
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C. Huang, G. T. De Sanctis, P. J. O'Brien, J. P. Mizgerd, D. S. Friend, J. M. Drazen, L. F. Brass, and R. L. Stevens Evaluation of the Substrate Specificity of Human Mast Cell Tryptase beta I and Demonstration of Its Importance in Bacterial Infections of the Lung J. Biol. Chem., July 6, 2001; 276(28): 26276 - 26284. [Abstract] [Full Text] [PDF]
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J. L. Harris, A. Niles, K. Burdick, M. Maffitt, B. J. Backes, J. A. Ellman, I. Kuntz, M. Haak-Frendscho, and C. S. Craik Definition of the Extended Substrate Specificity Determinants for beta -Tryptases I and II J. Biol. Chem., September 7, 2001; 276(37): 34941 - 34947. [Abstract] [Full Text] [PDF]
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G. W. Wong, S. Yasuda, M. S. Madhusudhan, L. Li, Y. Yang, S. A. Krilis, A. Sali, and R. L. Stevens Human Tryptase epsilon (PRSS22), a New Member of the Chromosome 16p13.3 Family of Human Serine Proteases Expressed in Airway Epithelial Cells J. Biol. Chem., December 28, 2001; 276(52): 49169 - 49182. [Abstract] [Full Text] [PDF]
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, untranslated; 
, prepro; 
,
catalytic domain; 
, transmembrane tail). Lines represent introns and
flanking regions. The location of codons encoding the His (H), Asp (D),
and Ser (S) catalytic triad residues common to serine proteases are
indicated. Each intron is labeled with its phase (0, I, or II). Precise
lengths of 
