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Originally published In Press as doi:10.1074/jbc.M308209200 on October 28, 2003

J. Biol. Chem., Vol. 279, Issue 4, 2438-2452, January 23, 2004
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Mouse Chromosome 17A3.3 Contains 13 Genes That Encode Functional Tryptic-like Serine Proteases with Distinct Tissue and Cell Expression Patterns*

Guang W. Wong{ddagger}§, Shinsuke Yasuda§, Nasa Morokawa, Lixin Li, and Richard L. Stevens

From the Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115

Received for publication, July 28, 2003 , and in revised form, October 15, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Probing of the mouse EST data base at GenBankTM with known tryptase cDNAs resulted in the identification of undiscovered serine protease transcripts whose genes reside at a 1.5-Mb complex on mouse chromosome 17A3.3. Mouse tryptase-5 (mT5), tryptase-6 (mT6), and mast cell protease-11 (mMCP-11) are new members of this serine protease superfamily whose amino acid sequences are 36–54% identical to each other and to their other 10 family members. The 13 functional mouse proteases can be subdivided into two subgroups based on conserved features in their propeptides. Of the three new serine proteases, mT6 is most widely expressed in tissues. mT5 is preferentially expressed in smooth muscle, whereas mMCP-11 is preferentially expressed in the spleen and bone marrow. In contrast to mT5 and mT6, mMCP-11 is also expressed in mast cells. Although mT6 and mMCP-11 are constitutively secreted when expressed in mammalian and insect cells, mT5 remains membrane-associated. The fact that recombinant mT5, mT6, and mMCP-11 possess non-identical expression patterns and substrate specificities suggests that each protease has a unique function in vivo. Of the 13 functional mouse tryptase genes identified at the complex, 12 have orthologs that reside in the syntenic region of human chromosome 16p13.3. The establishment of these ortholog pairs helps clarify the evolutionary relationship of the serine protease locus in the two species. This information provides a useful framework for the functional analysis of each protease using gene targeting and other molecular approaches.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The serine protease gene cluster at chromosome 16p13.3 contains the genes that encode human tryptase {alpha} (1), tryptase {beta}I (2), tryptase {beta}II (2, 3), tryptase {beta}III (2), transmembrane tryptase (TMT)1/tryptase {gamma} (4, 5), tryptase {delta} (6), tryptase {epsilon}/protease serine member S22 (PRSS22) (7), pancreasin/marapsin/PRSS27 (8), eosinophil serine protease-1 (Esp-1)/testisin/PRSS21 (9, 10), and EOS (11). There are five additional nonpeptidase homolog genes (currently designated as hSPL-2, -3, -4, -6, and -7) within the locus that probably encode non-functional proteins due to the presence of premature translation-termination codons (7). The subtelomeric region of human chromosome 16 where these genes reside is mutating at a rate ~300-fold faster than the rest of the genome in males (12). One explanation for this finding is that there is strong evolutionary pressure to expand some of the genes and delete others because of the respective beneficial and adverse roles.

The corresponding serine protease locus in the mouse genome resides at chromosome 17A3.3. When this study was initiated, 10 genes had been identified at the site that encode mouse mast cell protease (mMCP) 6 (13), mMCP-7 (14), mTMT (4), tryptase-4 (mT4)/mEsp-1/mTesp5/mTestisin/mPrss21 (15–17), testis serine protease-1 (mTessp1),2 distal intestinal serine protease (mDisp) (18), brain-specific serine protease-4 (mBssp-4)2/4733401N09Rik, pancreasin (8), implantation serine protease (mIsp) 1 (19), and mIsp-2 (20). Of these proteases, only mMCP-6, mMCP-7, and mT4 have been expressed and the recombinant proteases functionally characterized. Nevertheless, each member of this family of serine proteases appears to contain a unique substrate-binding cleft that also is more restricted than that of pancreatic trypsin. Screening of phage display peptide libraries and varied chromogenic substrates confirmed that recombinant mMCP-6 and mMCP-7 possess different substrate specificities (21, 22). hTryptases {alpha}, {beta}I, TMT/{gamma}, {delta}, and {epsilon} also have been shown to be functionally different (7, 23–27).

In regard to their in vivo function, administration of recombinant mMCP-6 or hTryptase {beta}I into the lungs significantly enhances the ability of MC-deficient W/Wv mice to combat a Klebsiella pneumoniae infection (24). This protective effect is mediated, in part, by the ability of both hTryptase {beta}I and mMCP-6 to selectively recruit large numbers of neutrophils (22, 24, 28). In support of these in vivo findings, Oh et al. (29) noted that neutrophil recruitment can be inhibited substantially in ovalbumin-treated mice by the tryptase tetramer inhibitor MOL-6131. Although the amino acid sequences of mMCP-6 (13) and mMCP-7 (14) are 71% identical, these two tryptases exhibit different biologic activities in vivo. When placed into the peritoneal cavities of mice, mMCP-7 preferentially induces the extravasation of eosinophils (24). The {alpha} chain of fibrinogen is a physiologic substrate of mMCP-7 (21). Thus, the latter mouse tryptase also appears to help prevent the formation of fibrin/platelet clots at inflammatory sites. Unlike mMCP-6 and mMCP-7, mTMT has a membrane-spanning domain at its C terminus that anchors this tryptase in the plasma membrane when MCs degranulate (4, 26). MCs physically contact T cells (30), and exposure of Jurkat T cells to recombinant hTMT results in altered gene expression (26). TMT therefore appears to be a novel granule mediator that mouse and human MCs use to preferentially regulate adjacent cell types that they physically contact. TMT can adversely affect lung function due to its ability to activate IL-13/IL-4R{alpha}/STAT6-dependent signaling pathways. It was discovered recently that pro-urokinase-type plasminogen activator is a preferred substrate of recombinant hTryptase {epsilon} (31).

The potential roles of the tryptase family of serine proteases in varied inflammatory disorders has prompted the pharmaceutical industry to begin generating low molecular weight protease inhibitors before the number of family members had been deduced. The ability to create inhibitors that selectively inactivate a harmful protease is dependent on our knowledge of how many related protease genes exist in humans, where and when they are expressed, and what are their beneficial and adverse roles in vivo. This knowledge becomes attainable, in part, with the generation of the complete sequence of the human genome and its transcripts. To deduce the function of these proteases in mouse models of human disease, it is imperative that their corresponding orthologs be identified. It is presently unclear how many functional tryptic genes of this family are present in the mouse genome. Even considering the known mouse protease genes on chromosome 17A3.3, it was difficult to assign unambiguously each one to its corresponding human ortholog. For this reason, it was not obvious which mouse protease studies would be relevant to humans. In the present study, we attempt to resolve these issues by comparative genome analysis of the mouse and human loci where these tryptic genes reside. In this process, we describe three new functionally distinct mouse tryptic proteases that are not coordinately expressed in vivo. We also establish that 12 mouse serine protease genes at chromosome 17A3.3 have human orthologs. This information provides a useful framework for the systematic functional analysis of each mouse/human protease pair using transgenic approaches.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Analysis of the Serine Protease Gene Cluster on Mouse Chromosome 17A3.3 and Cloning of mT5, mT6, and mMCP-11 cDNAs—The nucleotide sequences of the mMCP-6, mMCP-7, mT4, and mTMT transcripts were used as templates to search for novel, but related, mouse ESTs or genomic sequences in the varied data bases of GenBankTM. As noted under "Results," this approach resulted in the identification of three new members of the tryptase superfamily (designated as mT5, mT6, and mMCP-11). Based on the nucleotide sequences of the identified EST clones, the entire coding regions of the mT5, mT6, and mMCP-11 transcripts were obtained using cDNA libraries from BALB/c mouse skeletal muscle, spleen (Clontech, Palo Alto, CA), and IL-3-developed BALB/c mouse bone marrow-derived MCs (mBMMCs) (32), respectively. The oligonucleotides used in these PCRs were 5'-GCAGGTGTACTATGGAGCTGGCTCTG-3' and 5'-TTAGGGTCCCAGGAGGAAGAAGGCGCTACTG-3' for the mT5 transcript, 5'-CAATGAGGGGTGCTTCCCACCTCCAG-3' and 5'-GAGGCTCAGGCGAGCCTGGATCCAG-3' for the mT6 transcript, and 5'-GATATGTGCTTGGGGATGCTCTGG-3' and 5'-AGAAAGTGAGGCTGGACCAGAAGG-3' for the mMC-P-11 transcript. The resulting products were purified on 1% agarose gels, subcloned, and sequenced to confirm their identities.

The nucleotide sequences of the cDNAs that encode mT4, mT5, mT6, mMCP-6, mMCP-7, mMCP-11, mTMT, mTessp1, mDisp, mBssp-4, mPancreasin, mIsp-1, and mIsp-2 were aligned against the sequence of chromosome 17A3.3 (from the Mouse Genome Project) with the "blastn" algorithm available at the web site for the National Center for Biotechnology Information. The draft sequence of mouse chromosome 17A3.3 also was searched with the translated tblastn algorithm for Spl genes that encode conserved peptide sequences in serine proteases.

Transcript Analysis—A semi-quantitative PCR approach was used to screen multiple tissue cDNA libraries (Clontech) for the presence of the varied mouse tryptase transcripts. The oligonucleotides used in these transcript studies and the sizes of the resulting PCR products are summarized in Table I. Glyceraldehyde-3-phosphate dehydrogenase (G3PDH)-specific oligonucleotides (Clontech) were used as positive controls in these transcript profiling studies. MCs express mMCP-6 (13), mMCP-7 (14), and mTMT (4). Thus, total RNA was isolated from BALB/c, C57BL/6, 129Sv, and WBB6F1-W/Wv mBMMCs cultured in the presence of IL-3 for 1–5 weeks (32). At week 3, >99% of the cells in these cultures are MCs. A semi-quantitative reverse transcriptase-PCR approach (30–32 cycles) was then used to assess the relative levels of the mT5, mT6, and mMCP-11 transcripts in the different populations of MCs. As noted under "Results," IL-3-developed mBMMCs transiently express mMCP-11 mRNA. Thus, to investigate the influence of MC-regulatory cytokines on the expression of mMCP-11, additional experiments were carried out using BALB/c-derived mBMMCs that had been developed by culturing progenitors in the presence of IL-3 with varied combinations of IL-4, IL-9, IL-10, IL-12, or kit ligand as described in other MC studies (33–36). Whether or not two mouse MC lines (V3 and C1.MC/C57.1), two macrophage cell lines (RAW and WEHI-3), a T cell hybridoma (BY155), and a fibroblast cell line (mTc-1) contain mMCP-11 mRNA also was evaluated. The V3 and C1.MC/C57.1 cell lines have been maintained continuously for >1 decade. Thus, there is no contaminating cell type in these MC lines.


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  		TABLE I
Primer sets used to evaluate the expression of varied mouse tryptases in tissues

Indicated are the forward and reverse oligonucleotides used to evaluate the expression of the varied mouse tryptase transcripts in tissues, as well as the sizes of their PCR products.

 
Expression of Recombinant Mouse Proteases in Mammalian and Insect Cells—Bioengineered forms of recombinant mT6, mMCP-11, and mBssp-4 were generated in mammalian cells that contained the His6 and V5 (Gly-Lys-Pro-Ile-Pro-Asn-Pro-Leu-Leu-Gly-Leu-Asp-Ser-Thr) peptides at their C termini, as described previously for recombinant hTryptase {epsilon} (7). In each instance, the coding region of the tryptase cDNA was placed in the mammalian expression vector pcDNA3.1/V5-His TOPO (Invitrogen). Vector lacking an insert served as a negative control in the transfection experiments. mT4, mT5, mPancreasin, mDisp, and mTessp1 possess predicted glycosylphosphatidylinositol (GPI) signal sequences at their C termini. Thus, in these instances, the expressed protease contained the 8-mer FLAG (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) peptide inbetween the propeptide and the mature domain of the protease. African green monkey SV40-transformed kidney COS-7 cells (line CRL-1651; American Type Culture Collection (ATCC), Manassas, VA) and human embryonic kidney HEK 293T cells (line CRL-1573; ATCC) were cultured in DMEM containing 10% fetal calf serum. Transient transfections were performed in both cell types with SuperFect (Qiagen, Valencia, CA), according to the manufacturer's instructions. Cells were plated at a density of ~2 x 105 cells/well in 6-well plates 24 h before transfection. Twenty four h after transfection, the treated cells were washed and then cultured in serum-free Opti-MEM I medium for another 24 h before the conditioned medium and cell pellets were collected.

Recombinant mT5, mT6, and mMCP-11 also were generated in High Five insect cells using a modification of the expression system we developed previously to generate recombinant mMCP-6, mMCP-7 (21), mT4, and hTryptases {alpha}, {beta}I, {beta}II, TMT/{gamma}, and {epsilon}. Insect cells lack the post-translational machinery needed to remove the propeptide of a mammalian serine protease zymogen. Thus, the FLAG peptide was placed inbetween the natural propeptide and first residue of the catalytic domain to allow activation of the recombinant bioengineered zymogen by enterokinase. The FLAG peptide also facilitates the purification of the recombinant proteases from the conditioned medium using an immunoaffinity column. In this expression system, the C-terminal hydrophobic domain in mT5 was removed. Protease-expressing insect cells were cultured at room temperature in serum-free, X-press medium for 6–7 days. Generally, 750 ml of conditioned medium were loaded onto a freshly prepared 1-ml column containing anti-FLAG M2 antibody (Sigma). After each column was washed with 250 ml of Tris-buffered saline (pH 7.0), 0.1 M glycine (pH 3.5) was used to elute the bound protease. In each instance, 10 1-ml fractions were collected into tubes containing 20 µl of 1 M Tris-HCl (pH 8.0). Samples of the resulting fractions were analyzed by SDS-PAGE for the presence of Coomassie Blue-stained proteins and for immunoreactive tryptases by using anti-FLAG M2 antibody (Sigma). Protease-enriched fractions were pooled and their protein contents estimated using the micro BCA protein assay reagent kit (Pierce).

Evaluation of the Glycosylation Status and Substrate Specificities of Recombinant Mouse Proteases—Many mouse and human tryptases contain N-linked glycans (37–39), and expression/site-directed mutagenesis analysis of mMCP-7 revealed that these glycans often are important in thermal stability (40). Analysis of their predicted primary amino acid sequences revealed potential N-linked glycosylation sites in mT5, mMCP-11, mBssp-4, mTessp1, and mPancreasin. Thus, to determine whether any of these proteases contain N-linked glycans, a sample of the conditioned medium or cell lysate from each transfectant was incubated with PNGaseF (New England Biolabs, Beverly, MA) according to the manufacturer's suggested conditions. The resulting digests were then subjected to SDS-PAGE/immunoblot analysis.

The biotinylated D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone (PPACK) active-site assay developed by Williams et al. (41) was used to evaluate whether or not the varied uncharacterized mouse proteases in this study are enzymatically active when expressed in insect cells. Each purified zymogen was exposed to PPACK (Calbiochem) before and after the enterokinase activation step. The treated material was subjected to SDS-PAGE, the separated proteins transferred to a polyvinylidene difluoride membrane, and the resulting blot incubated with horseradish peroxidase-conjugated streptavidin (Bio-Rad).

The substrate specificities of recombinant mT5, mT6, and mMCP-11 were then compared in more detail by using the seven trypsin-susceptible chromogenic substrates t-butoxycarbonyl-Leu-Gly-Arg-pNA, benzyloxycarbonyl-Arg-Arg-pNA, H-D-Ile-Phe-Lys-pNA, H-D-Leu-Thr-Arg-pNA, H-Glu-Gly-Arg-pNA, 3-carboxy-propionyl (Suc)-Ala-Ala-Pro-Arg-pNA, (Bachem, King of Prussia, PA), and tosyl-Gly-Pro-Arg-pNA (Sigma). Purified insect cell-derived mT5, mT6, and mMCP-11 were activated as described previously for other recombinant tryptase pseudozymogens by incubating the purified recombinant protein for 2 h at 37 °C in 10 mM Tris-HCl buffer (pH 5.5) supplemented with 5 mM calcium chloride and ~0.4 units of enterokinase (New England Biolabs). One microliter of each chromogenic substrate (50 µg) was then added to 40 µl of 10 mM Tris-HCl buffer (pH 8.0) containing ~1 µg of activated recombinant tryptase. The proteolytic activity of the sample was measured as a change in optical density at 405 nm relative to that obtained with enterokinase-containing buffer. The digestion conditions were carried out in such a way that allowed us to evaluate the substrate preference of each protease for the seven examined substrates. mT5 and mT6 were incubated at room temperature with their substrates for 2 h (rather than 20 min) because in no instance was a large amount of substrate cleaved. As noted under "Results," mMCP-11 cleaved H-D-Leu-Thy-Arg-pNA as effectively as pancreatic bovine trypsin (Sigma). Thus, to ensure that the enzymatic reactions were linear during the chosen time frame, mMCP-11 was incubated at room temperature with its substrates for only 20 min. Each enzymatic assay was done in duplicate.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Isolation of mT5, mT6, mMCP-11 cDNAs, and Analysis of the Protease Gene Cluster on Mouse Chromosome 17A3.3 and Human Chromosome 16p13.3—When the cDNAs that encode mMCP-6, mMCP-7, mTMT, and mT4 were used as templates to screen the GenBankTM mouse EST data base at the start of this study in 2001, multiple truncated ESTs were identified that were somewhat homologous with one or more of the target cDNAs. The relevant ESTs were obtained from the I.M.A.G.E. consortium. Further nucleotide sequence analysis of their inserts revealed that they encoded novel mouse serine proteases (designated as mT5, mT6, and mMCP-11).3 Based on the sequences of these ESTs and their relevant genomic fragments, the entire coding regions of the mT5 (Fig. 1A), mT6 (Fig. 1B), and mMCP-11 (Fig. 1C) transcripts eventually were obtained from BALB/c mouse skeletal muscle, spleen, and mBMMCs, respectively. The full-length mT5, mT6, and mMCP-11 cDNAs consist of 1450, 1361, and 1215 nucleotides, respectively. Their 5'- and 3'-untranslated regions consist of 24 and 436 nucleotides for the mT5 transcript, 21 and 505 nucleotides for the mT6 transcript, and 99 and 159 nucleotides for the mMCP-11 transcript, respectively. The putative translation-initiation codons in the mT5, mT6, and mMCP-11 transcripts conform to those found in most eukaryotic transcripts.



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  		FIG. 1.
Cloning of mT5, mT6, and mMCP-11 cDNAs. Shown are the deduced nucleotide sequences of the mt5 (A), mt6 (B), and mMCP-11 (C) transcripts. The N-linked glycosylation site(s) (circled), components of the catalytic triad (solid boxes), translation-initiation site (*), translation-termination site (–), polyadenylation regulatory sequence (underline), hydrophobic signal peptide (single bracket), and propeptide (double bracket) are indicated. The C-terminal hydrophobic extension peptide in mT5 is boxed. Nucleotide numbering begins at the 5'-untranslated region of the isolated cDNA; amino acid numbering (within brackets at left) begins with the mature protein.

 



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  		FIG. 1.
Cloning of mT5, mT6, and mMCP-11 cDNAs. Shown are the deduced nucleotide sequences of the mt5 (A), mt6 (B), and mMCP-11 (C) transcripts. The N-linked glycosylation site(s) (circled), components of the catalytic triad (solid boxes), translation-initiation site (*), translation-termination site (–), polyadenylation regulatory sequence (underline), hydrophobic signal peptide (single bracket), and propeptide (double bracket) are indicated. The C-terminal hydrophobic extension peptide in mT5 is boxed. Nucleotide numbering begins at the 5'-untranslated region of the isolated cDNA; amino acid numbering (within brackets at left) begins with the mature protein.

 



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  		FIG. 1.
Cloning of mT5, mT6, and mMCP-11 cDNAs. Shown are the deduced nucleotide sequences of the mt5 (A), mt6 (B), and mMCP-11 (C) transcripts. The N-linked glycosylation site(s) (circled), components of the catalytic triad (solid boxes), translation-initiation site (*), translation-termination site (–), polyadenylation regulatory sequence (underline), hydrophobic signal peptide (single bracket), and propeptide (double bracket) are indicated. The C-terminal hydrophobic extension peptide in mT5 is boxed. Nucleotide numbering begins at the 5'-untranslated region of the isolated cDNA; amino acid numbering (within brackets at left) begins with the mature protein.

 
Analysis of the genomic contigs in GenBankTM revealed that the mT5, mT6, and mMCP-11 genes reside on chromosome 17A3.3 (see GenBankTM accession number NT_039649 [GenBank] ) within a 1.5-Mb sequence that contains the mMCP-6, mMCP-7, mT4, mTMT, mPancreasin, mBssp-4, mTessp1, mDisp, mIsp-1, and mIsp-2 genes (Fig. 2A). When this region of the C57BL/6 mouse genome was screened for nucleotide sequences that encode highly conserved amino acid sequences present in other serine proteases, a related gene (designated mSpl-1) was identified that resides inbetween the mIsp-2 and mMCP-7 genes (Fig. 2A). If expressed in C57BL/6 mice, the mSpl-1 gene would encode a protein that lacks enzymatic activity due to loss of critical residues that form the catalytic triad in serine proteases.



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  		FIG. 2.
Location of serine protease genes on mouse chromosome 17A3.3. A, diagram of the recently described genomic contig (accession number NT_039649 [GenBank] ) that spans a 1.5-Mb region of mouse chromosome 17A3.3. Shown are the relative positions and spacing of the 13 genes that encode the enzymatically active serine proteases within the cluster, as well as the mSpl-1 gene that encodes an enzymatically inactive protease. In each instance, the arrow above the gene denotes its orientation in the chromosome. Only the serine protease genes and their related family members are shown in this region of the chromosome. B, alignment of amino acid sequences within the propeptide regions of the group 1 (mT6, mTessp1, mT5, mT4, mDisp, mBssp-4, mPancreasin, and mTMT genes) and group 2 (mMCP-11, mIsp-1, mIsp-2, mSpl-1, mMCP-6, and mMCP-7) members. The conserved upstream Cys-Gly sequence and the conserved Arg/Lys at position –1 in each group 1 member are boxed. Also boxed is the conserved Gly at position –1 in each group 2 member. C, comparison of the exon/intron organization of the mT6, mTessp1, mT5, mT4, mDisp, mBssp-4, mPancreasin, mMCP-11, mIsp-1, mIsp-2, mMCP-7, mMCP-6, and mTMT genes. The size of each gene is indicated on the right. H, D, and S highlight where the codons reside that encode the catalytic triad amino acids in each serine protease.

 
Examination of the varied mouse tryptic proteases revealed conserved features in the propeptides that provide a convenient way to segregate them into two subfamilies. When translated, the eight group 1 tryptic proteases (i.e. mT4, mT5, mT6, mTessp1, mDisp, mBssp-4, mPancreasin, and mTMT) possess a conserved Cys in their propeptides (Fig. 2B). This Cys is predicted to form a disulfide bond with a conserved Cys that resides in the catalytic chain of the protease as shown previously (26) for recombinant hTMT. Thus, group 1 tryptic proteases are two-chained proteases when enzymatically active. The group 1 enzymes also possess an Arg or Lys at the C-terminal ends of their propeptides. In contrast, the six group 2 tryptic proteases (i.e. mMCP-6, mMCP-7, mMCP-11, mIsp-1, mIsp-2, and mSpl-1) contain a Gly at the C-terminal ends of their propeptides; they also lack the Cys residue in the propeptide. The average size of the group 1 tryptic genes is ~5 kb, whereas the average size of the group 2 tryptic genes is only ~2 kb. The mMCP-6 and the mPancreasin genes are the smallest and largest tryptic genes, respectively. The exon/intron organizations and the sizes of the chromosome 17A3.3 family of serine protease genes are depicted in Fig. 2C and at the GenBankTM sites. The distance that separates each tryptase gene on the chromosome ranges from 1.6 to 100 kb (Fig. 2A). However, the distance that separates the group 1 family of genes (except the mTMT gene) from the group 2 family of genes is 1.2 Mb.

Comparison of the mouse serine protease locus on chromosome 17A3.3 and its corresponding locus on the syntenic region of chromosome 16p13.3 revealed that the mouse locus contains 14 genes that encode 13 functional tryptic proteases and one Spl gene. The corresponding locus in the human genome contains 14 genes that encode 9 functional tryptic proteases and 5 SPL genes (Fig. 3). Twelve pairs of ortholog mouse/human serine protease genes can be established by pairwise protein and nucleotide sequence comparison. The amino acid sequence identity between each mouse/human ortholog ranges from 38 to 79%. The mT6, mTMT, mMCP-6, mMCP-7, mPancreasin, and mBssp-4 genes possess functional human orthologs. In contrast, the human genes that correspond to the mIsp-1, mIsp-2, mMCP-11, mDisp, and mTessp1 genes encode proteins that are not enzymatically active due to mutations that lead to premature translation-termination codons. Of the 13 functional mouse serine protease genes present on chromosome 17A3.3, the mT5 gene is the only one that does not have a discernible human counterpart in the human genome. Similarly, the hTryptase {alpha} gene does not appear to have a counterpart in the C57BL/6 mouse genome.



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  		FIG. 3.
Comparison of the mouse and human tryptic protease gene locus. The schematic diagram depicts all serine protease and SPL/Spl genes found on mouse chromosome 17A3.3 and human chromosome 16p13.3. The distance separating each protease gene is not drawn to scale. Ortholog pairs of functional mouse/human protease genes are connected by solid lines. The dashed lines link the indicated functional mouse gene with its hSPL gene. Percent amino acid identity between each mouse protease and its corresponding human ortholog is shown on the right. As of Dec. 16, 2003, the Human Genome Project still has not been able to assign the precise location of the hTryptase-{alpha} gene (GenBankTM Locus accession number 7176) on chromosome 16p13.3.

 
Expression of Mouse Proteases at the mRNA Level in Tissues and Cells—PCR analysis of multiple BALB/c mouse tissue cDNA panels revealed that the varied members of the chromosome 17A3.3 family of mouse serine proteases are differentially expressed in tissues (Fig. 4). Some proteases (e.g. mT6, mMCP-6, and mMCP-7) are expressed in numerous tissues. Others (e.g. mT4, mDisp, and mIsp-1) are more limited in their expression, as shown previously (4) for the mTMT transcript. Many of the mouse tryptases are regulated temporally during embryonic development. Some (e.g. mTessp1 and mT4) are not abundantly expressed in any stage of embryonic development, whereas others are selectively expressed either during the early (e.g. mIsp-1 and mIsp-2) or late (e.g. mMCP-6 and mMCP-11) stages. No mSpl-1 cDNA was identified in the GenBankTM EST data base. Moreover, the level of mSpl-1 mRNA was below detection in 20 different BALB/c mouse adult and fetal tissue cDNA libraries (data not shown). The accumulated data imply that the mSpl-1 gene is a nontranscribed pseudogene in the BALB/c mouse. Alternatively, its transcript is developmentally restricted or rapidly degraded.



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  		FIG. 4.
Expression profile of the varied members of the chromosome 17A3.3 family of mouse proteases in adult and embryonic mouse tissues. A semi-quantitative PCR approach was used to screen various BALB/c mouse cDNA libraries from Clontech to identify those tissues that contain the highest levels of mT4, mT5, mT6, mMCP-11, mMCP-6, mMCP-7, mTessp1, mDisp, mBssp-4, mPancreasin, mIsp-1, and mIsp-2 mRNA.

 
No MC population was identified that contained abundant amounts of mT5 or mT6 mRNA (data not shown). In contrast, mMCP-11 mRNA was abundant in mBMMCs of >99% purity generated from four mouse strains (Fig. 5A). mMCP-11 mRNA was detected in the V3 and C57.1 MC lines but not in a fibroblast cell line, a T cell hybridoma, or two macrophage lines (Fig. 5B). Thus, like the mMCP-6 and mMCP-7 transcripts, the mMCP-11 gene is expressed in MCs and their progenitors. Similar to the expression pattern of the mMCP-7 transcript in developing mBMMCs (14), the level of the mMCP-11 transcript in these cells decreased substantially after 3 weeks of culture in IL-3-enriched medium (Fig. 5A). The MC-regulatory cytokines IL-4, IL-9, IL-10, IL-12, and kit ligand were unable to affect dramatically the levels of mMCP-11 mRNA in IL-3-developed mBMMCs (data not shown).



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  		FIG. 5.
Expression of the mMCP-11 transcript in IL-3-developed mBMMCs and various mouse cell lines. A, mMCP-11 mRNA expression in mBMMCs was evaluated using an reverse transcriptase-PCR approach. Total RNA was isolated from BALB/c, C57BL/6, 129Sv, and WBB6F1-W/Wv mBMMCs cultured in medium supplemented with recombinant IL-3 for 1, 3, and 5 weeks. The cDNAs that were generated were subjected to 30 cycles of PCR (upper panel). Expression of G3PDH was evaluated by 25 cycles of PCR (lower panel). For negative controls, PCRs were carried out in the absence of a cDNA. B, two MC lines (V3 and C1.MC/C57.1), two macrophage cell lines (RAW and WEHI-3), a T cell hybridoma (BY155), and a fibroblast cell line (mTc-1) also were evaluated for their expression of the mMCP-11 (upper panel) and G3PDH (lower panel) transcripts.

 
Analysis of the Mouse Chromosome 17A3.3 Family of Serine Proteases at the Protein Level—The amino acid sequences of the chromosome 17A3.3 family of mouse proteases are 31–71% identical to each other (Table II). The prepropeptides of mT5, mT6, and mMCP-11 consist of 54, 33, and 34 amino acids, respectively (Fig. 1, Fig. 1, Fig. 1). All mature members of the family except mT5 and mPancreasin have an N-terminal sequence of Ile-Val-Gly-Gly (Fig. 6A). mT5 differs in that it has a Ser at residue 3, whereas mPancreasin has a Met at residue 1. Like some members of its family, mT5 possesses a putative GPI signal sequence. It also has a 27-mer hydrophobic domain at its C terminus. In their mature forms, the 26–31-kDa catalytic domains of mT5, mT6, and mMCP-11 possess 10, 10, and 11 Cys residues, respectively. Cys–9 within the propeptides of mT5 and mT6 is predicted to form a disulfide bond with Cys112 and Cys110 in their respective catalytic main chains. Phylogenetic analysis of the varied mouse tryptases is shown in Fig. 6B. cDNAs that appear to be the rat orthologs of mT4, mT5, mT6, mMCP-6, mMCP-7, mMCP-11, mTMT, mBssp-4, mIsp-1, mIsp-2, and mTessp1 have been deposited in GenBankTM. Although no rat cDNAs that correspond to mPancreasin and mDisp have been described, examination of a rat chromosome 10q12-derived contig (GenBankTM accession number NW_042648) revealed that the rat genome possesses genes that correspond to the latter two mouse tryptase genes (data not shown). Moreover, the rat tryptase locus on chromosome 10q12 contains three additional genes (accession numbers XP_220241, XP_220242, and XP_220250) that encode serine proteases that are most homologous to mIsp-1 and mIsp-2 (Fig. 6B). Thus, of the three species examined, the tryptase locus on rat chromosome 10q12 appears to harbor the greatest number of functional protease genes.


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  		TABLE II
Comparison of the amino acid sequences of the chromosome 17A3.3 family of mouse tryptases

The percent identities are indicated.

 



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  		FIG. 6.
Comparison of the amino acid of sequences of the tryptase family of serine proteases present in mice and other mammals. A, the amino acid sequences of the mature forms of the chromosome 17A3.3 family of mouse proteases were aligned using the PILEUP program of the "GCG" software package. Identical amino acids are shaded. Numbering (left) begins at the first residue in the mature portion of each protease. The seven loops (designated A, B, C, D, 1, 2, and 3) predicted to form the substrate-binding cleft of each serine protease are indicated (bars). The arrows point to the conserved Cys residue found in the catalytic main chain of each group 1 mouse tryptase that forms a disulfide bond with the conserved Cys residue found in the propeptide. B, the amino acid sequences of the tryptases noted above were extracted from the GenBankTM data base. A cladogram was then generated by the TreeView program. The GenBankTM accession numbers for the depicted proteases are as follows: hEsp-1/testisin/PRSS21 (AB031329 [GenBank] ), hPancreasin/hMarapsin (AJ306593 [GenBank] ), hTryptase {beta}I (NP_003294), hTryptase {beta}II (NP_024164), hTryptase {beta}III (C35863 [GenBank] ), hTryptase {alpha} (NP_003293 [GenBank] ), hTMT/hTryptase {gamma} (AF175522 [GenBank] ), hTryptase {delta} (NP_036349 [GenBank] ), hTryptase {epsilon}/PRSS22 (NP_071402 [GenBank] ), hEOS (NP_690851 [GenBank] ), hTrypsin (pancreas; NP_002769 [GenBank] ); mTessp1 (BAB68561 [GenBank] , mT4/mEsp-1/mPrss21 (NP_065233 [GenBank] ), mT5 (AAP23216 [GenBank] and Fig. 1A), mT6 (AY262280 [GenBank] and Fig. 1B), mT6 (AY262280 [GenBank] ), mDisp (AJ243866 [GenBank] ), mBssp-4/4733401N09Rik (AB010778 [GenBank] ), mPancreasin (NP_780649 [GenBank] ), mMCP-11 (AY261775 [GenBank] and Fig. 1C), mIsp-1 (NP_444489 [GenBank] ), mIsp-2 (NP_444490 [GenBank] ), mMCP-7 (AAA39992 [GenBank] , mMCP-6 (P21845 [GenBank] ), mTMT/mTryptase {gamma} (NP_036164 [GenBank] ), mProstasin (NP_579929 [GenBank] ), rT6 (XP_220208 [GenBank] ), rTessp1 (XP_220208 [GenBank] ), rT5 (XP_220209 [GenBank] ), rEsp-1 (BAC57949 [GenBank] , rDisp (predicted from rat contig NW_042648), rBssp-4 (XP_220222 [GenBank] ), rPancreasin (predicted from rat contig NW_042648), rT7/rMCP-11 (XP_220247 [GenBank] ), rIsp-1 (XP_220248), rIsp-2 (XP_220249), rMCP-7 (P27435 [GenBank] ), rMCP-6 (P50343 [GenBank] ), rTMT/rTryptase {gamma} (AY196208 [GenBank] ), rat XP_220241 (XP_220241), rat XP_220242 (XP_220242), rat XP_220250 (XP_220250); bovine tryptase-1 (CAA64438 [GenBank] , bovine tryptase-2 (NP_776627 [GenBank] ); sheep tryptase-1(CAB41988 [GenBank] , sheep tryptase-2 (Q9XSM2); porcine MC tryptase (BAA93614 [GenBank] , porcine tryptase TC30 (BAB85761 [GenBank] ; dog tryptase (AAA30854 [GenBank] , dog mastin/dMCP-3 (P19236 [GenBank] ); gerbil tryptase (S56160 [GenBank] ); and horse (equine) tryptase-1 (CAD56807 [GenBank] .

 



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  		FIG. 6.
Comparison of the amino acid of sequences of the tryptase family of serine proteases present in mice and other mammals. A, the amino acid sequences of the mature forms of the chromosome 17A3.3 family of mouse proteases were aligned using the PILEUP program of the "GCG" software package. Identical amino acids are shaded. Numbering (left) begins at the first residue in the mature portion of each protease. The seven loops (designated A, B, C, D, 1, 2, and 3) predicted to form the substrate-binding cleft of each serine protease are indicated (bars). The arrows point to the conserved Cys residue found in the catalytic main chain of each group 1 mouse tryptase that forms a disulfide bond with the conserved Cys residue found in the propeptide. B, the amino acid sequences of the tryptases noted above were extracted from the GenBankTM data base. A cladogram was then generated by the TreeView program. The GenBankTM accession numbers for the depicted proteases are as follows: hEsp-1/testisin/PRSS21 (AB031329 [GenBank] ), hPancreasin/hMarapsin (AJ306593 [GenBank] ), hTryptase {beta}I (NP_003294), hTryptase {beta}II (NP_024164), hTryptase {beta}III (C35863 [GenBank] ), hTryptase {alpha} (NP_003293 [GenBank] ), hTMT/hTryptase {gamma} (AF175522 [GenBank] ), hTryptase {delta} (NP_036349 [GenBank] ), hTryptase {epsilon}/PRSS22 (NP_071402 [GenBank] ), hEOS (NP_690851 [GenBank] ), hTrypsin (pancreas; NP_002769 [GenBank] ); mTessp1 (BAB68561 [GenBank] , mT4/mEsp-1/mPrss21 (NP_065233 [GenBank] ), mT5 (AAP23216 [GenBank] and Fig. 1A), mT6 (AY262280 [GenBank] and Fig. 1B), mT6 (AY262280 [GenBank] ), mDisp (AJ243866 [GenBank] ), mBssp-4/4733401N09Rik (AB010778 [GenBank] ), mPancreasin (NP_780649 [GenBank] ), mMCP-11 (AY261775 [GenBank] and Fig. 1C), mIsp-1 (NP_444489 [GenBank] ), mIsp-2 (NP_444490 [GenBank] ), mMCP-7 (AAA39992 [GenBank] , mMCP-6 (P21845 [GenBank] ), mTMT/mTryptase {gamma} (NP_036164 [GenBank] ), mProstasin (NP_579929 [GenBank] ), rT6 (XP_220208 [GenBank] ), rTessp1 (XP_220208 [GenBank] ), rT5 (XP_220209 [GenBank] ), rEsp-1 (BAC57949 [GenBank] , rDisp (predicted from rat contig NW_042648), rBssp-4 (XP_220222 [GenBank] ), rPancreasin (predicted from rat contig NW_042648), rT7/rMCP-11 (XP_220247 [GenBank] ), rIsp-1 (XP_220248), rIsp-2 (XP_220249), rMCP-7 (P27435 [GenBank] ), rMCP-6 (P50343 [GenBank] ), rTMT/rTryptase {gamma} (AY196208 [GenBank] ), rat XP_220241 (XP_220241), rat XP_220242 (XP_220242), rat XP_220250 (XP_220250); bovine tryptase-1 (CAA64438 [GenBank] , bovine tryptase-2 (NP_776627 [GenBank] ); sheep tryptase-1(CAB41988 [GenBank] , sheep tryptase-2 (Q9XSM2); porcine MC tryptase (BAA93614 [GenBank] , porcine tryptase TC30 (BAB85761 [GenBank] ; dog tryptase (AAA30854 [GenBank] , dog mastin/dMCP-3 (P19236 [GenBank] ); gerbil tryptase (S56160 [GenBank] ); and horse (equine) tryptase-1 (CAD56807 [GenBank] .

 
When transfected into COS-7 or HEK 293T cells, mT6, mMCP-11, and mBssp-4 were constitutively secreted into the conditioned medium (Fig. 7A). In contrast, mT5, mPancreasin, mT4, mTessp1, and mDisp preferentially remained cell-associated (Fig. 7A). On the basis of the deduced amino acid sequences of their cDNAs, mT5 (Fig. 1A) and mMCP-11 (Fig. 1C) contain N-linked glycosylation sites like mPancreasin, mTessp1, and mBssp-4. As assessed by SDS-PAGE analysis, all five recombinant tryptases were reduced in size after PNGaseF treatment (Fig. 7B). Thus, the potential N-linked glycosylation sites are utilized. mT6 lacks an N-glycan site (Fig. 1B). As expected, recombinant mT6 was not susceptible to PNGaseF (Fig. 7B).



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  		FIG. 7.
Expression of recombinant mouse tryptic proteases in COS-7 and HEK 293T cells. A, COS-7 and HEK 293T cells were transfected with expression vector alone or expression vector containing an insert that encodes a bioengineered form of mT6, mT5, mMCP-11, mPancreasin, mBssp-4, mT4, mTessp1, or mDisp. Forty eight h later, samples of the resulting serum-free conditioned medium/supernatants (S) and lysates of the cell pellets (P) were evaluated for the presence of recombinant mBssp-4 and mPancreasin using anti-V5 antibody or the presence of mT5, mT6, mMCP-11, mDisp, and mTessp1 by using anti-FLAG antibody. Optimal levels of recombinant mT4, mT6, mMCP-11, and mDisp were obtained in HEK 293T cells, whereas optimal levels of recombinant mT5, mPancreasin, mBssp-4, and mTessp1 were obtained in COS-7 cells. The prominent low molecular weight bands obtained in the depicted mT4 and mT6 transfectants were not detected in a second set of transfections. The mT4- and mT6-derived fragments were not characterized. However, they probably are the result of nonspecific proteolysis of the exogenous zymogens in the HEK 293T cell expression system because low molecular weight fragments were not obtained when mT4 was expressed in COS-7 cells (15). B, the various tryptases were exposed to PNGaseF to evaluate whether or not they contained N-linked glycans. Conditioned medium was used for mT6, mMCP-11, and mBssp-4; cell lysates were used for mT5, mPancreasin, and mTessp1.

 
Comparison of the Substrate Specificities of the Mouse Chromosome 17A3.3 Family of Serine Proteases—When expressed in High Five insect cells, recombinant pro-mT5, pro-mT6, and pro-mMCP-11 could be quickly purified from the serum-free conditioned medium using anti-FLAG immunoaffinity columns (Fig. 8, A and B). Insect cells constitutively secreted recombinant pro-mT5 due to the fact that its expression construct encoded a truncated isoform that lacks the C-terminal hydrophobic domain. After enterokinase treatment, each zymogen was converted into an active protease that avidly bound PPACK (Fig. 8C). By using varied chromogenic substrates, the substrate specificities of recombinant mT5, mT6, and mMCP-11 were found to be nonidentical (Fig. 8D). As assessed by SDS-PAGE analysis, neither recombinant mT5, mT6, nor mMCP-11 underwent autolysis when the enzymatically active protease was incubated an additional 2 h at room temperature.



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  		FIG. 8.
Expression, purification, and enzymatic activity of insect cell-derived recombinant mT5, mT6, and mMCP-11. Pseudozymogen forms of pro-mT5, pro-mT6, and pro-mMCP-11 were expressed in insect cells that contained the FLAG peptide inbetween the natural propeptide and the catalytic portion of the mature tryptase. A, the purified zymogens were subjected to SDS-PAGE, and the resulting gel was stained with Coomassie Blue in order to evaluate the purity of each preparation. Molecular weight markers are shown on the left. B, blots, prepared from replicate gels, were probed with the anti-FLAG M2 antibody in order to show that in each instance the insect cell produced the appropriately sized product. C, each purified recombinant zymogen was then exposed to PPACK before (–) and after (+) enterokinase treatment to evaluate whether or not the expressed protease is biologically active. D, the substrate preferences of the three tryptases were compared in more detail with use of seven trypsin-susceptible chromogenic substrates. mT5, mT6, and mMCP-11 were incubated at room temperature with their substrates for 2 h, 2 h, and 20 min, respectively. Shown are the mean values (± range) obtained from an experiment done in duplicate.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
mT5 (Fig. 1A), mT6 (Fig. 1B), and mMCP-11 (Fig. 1C) represent new members of the tryptic family of serine proteases whose genes reside in the 1.5-Mb region of mouse chromosome 17A3.3 that contains the additional genes that encode mT4, mMCP-6, mMCP-7, mTMT, mDisp, mBssp-4, mTessp1, mPancreasin, mIsp-1, and mIsp-2 (Fig. 2A). With the identification of 13 functional protease genes at the complex, chromosome 17A3.3 contains the second largest cluster of serine protease genes in the mouse genome.

On the basis of conserved features within their propeptides, the chromosome 17A3.3 family of proteases can be subdivided into two groups (Fig. 2B). mT4, mT5, mT6, mTessp1, mDisp, mBssp-4, mPancreasin, and mTMT are group 1 proteases, whereas mMCP-11, mIsp-1, mIsp-2, mMCP-6, and mMCP-7 are group 2 proteases. All group 1 members are presumed to be two-chained serine proteases due to a disulfide bond that links the propeptide and catalytic domain. Other two-chained serine proteases include prostasin, hepsin, matriptase-1/MT-SP1, matriptase-2, human airway trypsin/HAT, urokinase-type plasminogen activator, tissue-type plasminogen activator, and plasminogen. In these latter proteases, the Cys residue in the propeptide resides 8–14 amino acids upstream of the cleavage site that results in the enzymatically active protease. A Gly always resides C-terminal of the conserved Cys residue. Both features are present in the group 1 family of chromosome 17A3.3 proteases (Fig. 2B). The tethered propeptide presumably is needed to prevent premature activation of each group 1 zymogen and to control its folding and/or targeting. However, it is also conceivable that the tethered propeptide serves as a recognition motif for the cognate substrate of the protease or receptor as occurs with the propeptide of urokinase-type plasminogen activator. All group 2 members lack the conserved Cys residue found in the propeptide of group 1 members (Fig. 2B). Thus, in their mature forms, the group 2 members are presumed to be single-chained enzymes.

Serine proteases are synthesized as zymogens that require a post-translational processing step to become enzymatically active. Often, this proteolytic activation event occurs at a trypsin-susceptible site located just before the N-terminal "Ile-Val-Gly-Gly" sequence present in the mature protease. The maturation process allows the newly formed N-terminal Ile to insert into the activation groove, which brings about an essential structural change in the protease. All group 2 members of the chromosome 17A3.3 family of tryptic proteases possess a Gly at the end of the propeptide instead of the Arg/Lys residue found at the corresponding position in group 1 enzymes (Fig. 2B). These data imply that the groups 1 and 2 proteases are activated via different mechanisms. hTryptase {epsilon} possesses an Arg at the C-terminal end of its propeptide (7). The recent finding that wild-type recombinant hTryptase {epsilon} but not its R-1A mutant autoactivates (31) raises the possibility that some group 1 mouse proteases also self-activate.

Although the distance that separates each protease gene within the group 1 or group 2 family is <100 kb (Fig. 2A), the distance that separates the two subgroups of protease genes is 1.2 Mb. The group 1 gene that encodes mTMT resides 3' of the group 2 gene that encodes mMCP-6 rather than 3' of the group 1 gene that encodes mPancreasin. Coupled with the data noted in Fig. 3, a chromosomal break apparently occurred in the tryptase locus downstream of the mPancreasin gene. This break was followed by an inversion of the genomic fragment that contained all group 2 protease genes as well as the one group 1 gene that encodes mTMT.

There are 13 functional tryptase genes and the mSpl-1 gene on mouse chromosome 17A3.3. Preliminary analysis of the corresponding locus on rat chromosome 10q12 revealed 16 serine protease genes, 13 of which encode the orthologs of the mouse genes noted in Fig. 2. The three additional tryptase genes in the rat genome most closely resemble the mIsp-2 gene (Fig. 6B). It is likely that the three additional rat genes came into existence after the evolutionary divergence of rats and mice via a gene duplication mechanism of the mIsp-1 or mIsp-2 gene. The corresponding human protease locus on the syntenic region of chromosome 16p13.3 contains more SPL genes (Fig. 3). mTMT, mMCP-6, mMCP-7, mPancreasin, mBssp-4, and mT6 have functional human orthologs. However, the genes that correspond to the mIsp-1, mIsp-2, mMCP-11, mDisp, and mTessp1 genes in the human genome possess premature translation-termination codons. The fact that the human genes that correspond to the latter five mouse protease genes no longer encode enzymatically active enzymes raises the possibility that they encoded deleterious proteins in our ancestors. Alternatively, some of the mouse enzymes are redundant or only needed in rodents. Although the rat genome contains an ortholog of the mT5 gene (Fig. 6B), a comparable gene is not present in the human genome (Fig. 3). Presumably, the mT5 gene evolved after the divergence of rodents and humans >70 million years ago. The fact that the mouse and rat genomes contain more functional tryptase genes than the human genome raises the possibility that some rodent tryptases possess overlapping substrate specificities. The presence of greater numbers of tryptases in rodents impacts the use of the mouse or rat to understand the role of human tryptases in various diseases. For example, in some instances multiple mouse tryptase genes may have to be inactivated to understand the function of a single human tryptase.

Most members of the chromosome 17A3.3 family of mouse proteases are not coordinately expressed. In addition, their expression often is restricted to a particular cell type or tissue. For example, we noted previously that mTMT is preferentially expressed in the intestines of BALB/c and C57BL/6 mice (4). With regard to the three new tryptases, mT6 is widely expressed in tissues (Fig. 4). In contrast, mT5 is preferentially expressed in the smooth muscle; mMCP-11 is preferentially expressed in the spleen and bone marrow. Many of the mouse proteases are expressed in the testis and/or prostate. The subtelomeric region of human chromosome 16 where the corresponding human protease genes reside is an intense male-specific recombination hotspot in the human genome (12). The expression of multiple members of the chromosome 17A3.3 family of proteases in the mouse testis and/or prostate suggests one or more of them play essential roles in male gonad development and function. Many of these proteases also are differentially expressed during embryonic development.

mMCP-11 is the fourth member of the tryptase family that is expressed in MCs and their progenitors (Fig. 5). mMCP-11 mRNA was detected in IL-3-developed mBMMCs and two mouse MC lines. However, similar to what occurs with mMCP-7 mRNA levels in mBMMCs, the level of the mMCP-11 transcript falls rapidly after 3 weeks of culture of the MCs in IL-3-enriched medium. The fact that investigators often use 6-week-old mBMMCs in their studies is likely the reason why the mMCP-11 transcript was missed in earlier transcript characterization studies of this MC population. IL-4, IL-9, IL-10, IL-12, and kit ligand had no dramatic effect on the steady-state levels of the mMCP-11 transcript in IL-3-developed mBMMCs. Thus, another MC-regulatory factor is needed to maintain high levels of expression of mMCP-11 mRNA in mature MCs. Alternatively, IL-3 dominantly inhibits the expression of this tryptase in MCs as occurs for the mMCP-1, mMCP-2, and mMCP-4 transcripts (42).

In 1998, Mitsui and co-workers isolated a cDNA they designated as mouse brain-specific serine protease-4 (mBssp-4). Despite its name, only small amounts of mBssp-4 mRNA are present in the brains of adult BALB/c mice (Fig. 4). At the nucleotide and amino acid levels, mBssp-4 is most similar to hTryptase {epsilon}/PRSS22 (Fig. 3), which is preferentially expressed in human epithelium (7). As found previously with the hTryptase {epsilon} transcript (7), epithelium-rich tissues such as the lung and eye contain substantial amounts of mBssp-4 mRNA (Fig. 4). The accumulated data strongly imply that mBssp-4 is the mouse ortholog of hTryptase {epsilon}. In support of this conclusion, a mouse epithelium-derived cDNA was recently deposited in GenBankTM (see accession number AK014645 [GenBank] ) that is identical to the mBssp-4 transcript. The discovery of the mouse ortholog of hTryptase {epsilon} now allows investigators the opportunity to answer questions pertaining to the function and regulation of hTryptase {epsilon} in mouse disease models.

Interrogation of the EST data base and PCR analysis from 20 different mouse tissues failed to detect the presence of transcripts that encode mSpl-1 (data not shown). Thus, it is presently unclear whether or not the mSpl-1 gene is transcribed. Azurocidin/CAP37/HBP is a neutrophil granule protein that exhibits potent antibiotic activity against Gram-negative bacteria (43, 44); it also regulates monocyte/macrophage chemotaxis, survival, and differentiation (45). Although azurocidin is 44% identical to human neutrophil elastase, it is not enzymatically active due to mutations in two of the three residues that constitute the "charge-relay system" in other serine proteases (46). The mSpl-1 gene cannot encode an enzymatically active serine protease due to the absence of the critical His, Asp, and Ser residues. Nevertheless, based on the azurocidin example, the mSpl-1 and hSPL-2, -3, -4, -6, and -7 genes could encode functional proteins that simply have lost their enzymatic activities.

At the protein level, the amino acid sequences of the varied members of the chromosome 17A3.3 family of mouse proteases are 36–71% identical (Table II). mT5 and mMCP-11 contain N-linked glycans but not mT6 (Fig. 7). The overall three-dimensional structures of the catalytic domains of mT5, mT6, and mMCP-11 are predicted to be similar to those of other serine proteases. For example, mT5, mT6, and mMCP-11 contain the residues that form the critical Trp-rich domain on the surface of other enzymatically active members of the family (40, 47). Mouse and human TMT/tryptase {gamma} remain anchored to the plasma membrane when MCs degranulate due to their C-terminal membrane-spanning domains (26). Of the 13 functional members of the chromosome 17A3.3 family of proteases, 6 of them (i.e. mT4, mT5, mPancreasin, mDisp, mTessp1, and mTMT) possess hydrophobic extensions at their C termini. It remains to be determined whether or not these tryptases reach the outer leaflet of the plasma membrane like mTMT and hTMT (26). Nevertheless, the fact that TMT remains cell-associated when MCs are activated implies that the C-terminal hydrophobic domain or a covalently linked GPI anchor is used to retain mT4, mT5, mPancreasin, mDisp, mTessp1, and mTMT in their appropriate membrane compartments.

The amino acid sequence of mMCP-11 is 61% identical to that of dog mastin/dMCP-3 (Fig. 6B). dMCP-3 differs from other characterized tryptases in that it dimerizes due to the presence of unpaired Cys75. Analysis of the deduced amino acid sequence of mMCP-11 (Figs. 1C and 6A) revealed the presence of 11 Cys residues. Cys32, Cys48, Cys131, Cys164, Cys187, Cys196, Cys206, and Cys224 in mMCP-11 correspond to the 8 Cys residues in hTryptase {beta}II that form 4 disulfide bonds (48). mMCP-11 differs from hTryptase {beta}II in that it has three additional Cys residues at positions 5, 210, and 245. Cys5 is located immediately downstream of the N terminus Ile-Val-Gly-Gly sequence of mature mMCP-11. This Cys residue is predicted to form an additional disulfide bond with either Cys210 or Cys245 located in the catalytic main chain. The presence of the remaining unpaired Cys raises the possibility that mMCP-11 dimerizes like dMCP-3.

mMCP-6 and mMCP-7 are the only two mouse tryptases that have been extensively characterized. Despite their overall 71% amino acid sequence identity, the substrate specificities of these two mouse tryptases are very different (21, 22). Serine proteases with tryptic-like specificity possess highly conserved amino acids such as Asp189, Gly216, and Gly226 (based on chymotrypsin numbering). Asp189 is located at the base of the substrate-binding pocket, and it interacts with the positively charged Lys or Arg P1 residue of the substrate. The small Gly at positions 216 and 226 within the substrate-binding pocket allow the bulky Lys/Arg P1 residue to fit into the substrate-binding pocket. The corresponding Asp189, Gly216, and Gly226 residues are present in mT5, mT6, and mMCP-11 (Figs. 1, 1, 1 and 6A). Because these data suggested that the three new mouse serine proteases possess tryptic-like activity, their substrate specificities were compared. The trypsin substrate tosyl-Gly-Pro-Arg-pNA was cleaved by all three mouse tryptases (Fig. 8D). However, recombinant mMCP-11 cleaved H-D-Leu-Thr-Arg-pNA more effectively than H-Glu-Gly-Arg-pNA. In contrast, recombinant mT5 cleaved H-Glu-Gly-Arg-pNA more effectively than H-D-Leu-Thr-Arg-pNA. Amino acid sequence alignment data (Fig. 6A) predict that the different enzymatic activities of mT5, mT6, and mMCP-11 are caused primarily by residue changes in loops A, C, and three that constitute the substrate-binding cleft of each protease. The physiologic substrates of the three new mouse tryptases remain to be determined. Nevertheless, our data suggest that two primordial serine protease genes at chromosome 17A3.3 duplicated repeatedly during evolution to give rise to multiple tryptic-like serine proteases in the mouse that possess distinct tissue distributions and substrate specificities.


   FOOTNOTES
 
* This work was supported in part by Grants HL-36110 and HL-63284 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This 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/EBI Data Bank with accession number(s) AAP23216 [GenBank] AY266139 [GenBank] , AAP20885 [GenBank] AY262280 [GenBank] , AAP21675 [GenBank] and AY261775 [GenBank] . The Mouse Genome Project has tentative designated mT5, mT6, and mMCP-11 as Prss32, Prss33, and Prss34 at GenBankTM Locus identification sites 69814, 353130, and 328780, respectively. Back

{ddagger} Present address: Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, MA 02142. Back

§ Both authors contributed equally to this work. Back

To whom correspondence should be addressed: Dept. of Medicine, Brigham and Women's Hospital, Smith Bldg., Rm. 616B, 1 Jimmy Fund Way, Boston, MA 02115. Tel.: 617-525-1231; Fax: 617-525-1310; E-mail: rstevens{at}rics.bwh.harvard.edu.

1 The abbreviations used are: TMT, transmembrane tryptase; Bssp-4, brain-specific serine protease-4; Disp, distal intestinal serine protease; Esp-1, eosinophil serine protease-1; G3PDH, glyceraldehyde-3-phosphate dehydrogenase; GPI, glycosylphosphatidylinositol; IL, interleukin; MC, mast cell; Isp, implantation serine protease; mMCP, mouse MC protease; mT4, mouse tryptase-4; mT5, mouse tryptase-5; mT6, mouse tryptase-6; PPACK, D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone; PRSS, protease serine member S; Tessp1, testis serine protease-1; PNGaseF, peptide:N-glycosidase; pNA, p-nitroanilide; mBMMCs, mouse bone marrow-derived MCs. Back

2 Although mBssp-4 and mTessp1 cDNAs have not been described in any scientific publication at the time of submission of this paper, their nucleotide sequences have the GenBankTM accession numbers BAB20262 [GenBank] and BAB68561 [GenBank] respectively. Back

3 The nucleotide and amino acid sequences of the three new mouse tryptases reported in this paper (GenBankTM/EBI Data Bank accession numbers AAP23216 [GenBank] and AY266139 [GenBank] for mT5, AAP20885 [GenBank] and AY262280 [GenBank] for mT6, and AAP21675 [GenBank] and AY261775 [GenBank] for mMCP-11) were released to the public in May 2003. No mT5, mT6, or mMCP-11 full-length cDNAs were present in the data bases when this work was initiated in 2001. An mT6-like cDNA still has not been isolated by another group. Nevertheless, the mT5 and mMCP-11 cDNAs described in our study resemble the recently released RIKEN cDNAs 2010001 (accession number BC024903 [GenBank] ) and C130091P12 (accession number AK081986 [GenBank] ), respectively. There is no journal publication relating to the latter two RIKEN cDNAs. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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