help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology, doi:10.1210/en.2003-0930
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Hansen, I. A.
Right arrow Articles by Allolio, B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hansen, I. A.
Right arrow Articles by Allolio, B.
Endocrinology Vol. 145, No. 4 1898-1905
Copyright © 2004 by The Endocrine Society

The Adrenal Secretory Serine Protease AsP Is a Short Secretory Isoform of the Transmembrane Airway Trypsin-Like Protease

Immo A. Hansen, Martin Fassnacht, Stefanie Hahner, Fabian Hammer, Markus Schammann, Susanne R. Meyer, Andrew B. Bicknell and Bruno Allolio

Endocrinology and Diabetes Unit (I.A.H., M.F., S.H., F.H., M.S., S.R.M., B.A.), Department of Medicine, University of Wuerzburg, D-97080 Wuerzburg, Germany; and School of Animal and Microbial Sciences (A.B.B.), The University of Reading, Whiteknights, Reading, Berkshire RG6 6AJ, United Kingdom

Address all correspondence and requests for reprints to: Immo A. Hansen, Department of Entomology, Watkins Drive, University of California, Riverside, California 92521. E-mail: immoh{at}citrus.ucr.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
To further elucidate the role of proteases capable of cleaving N-terminal proopiomelanocortin (N-POMC)-derived peptides, we have cloned two cDNAs encoding isoforms of the airway trypsin-like protease (AT) from mouse (MAT) and rat (RAT), respectively. The open reading frames comprise 417 amino acids (aa) and 279 aa. The mouse AT gene was located at chromosome 5E1 and contains 10 exons. The longer isoform, which we designated MAT1 and RAT1, has a simple type II transmembrane protein structure, consisting of a short cytoplasmic domain, a transmembrane domain, a SEA (63-kDa sea urchin sperm protein, enteropeptidase, agrin) module, and a serine protease domain. The human homolog of MAT1 and RAT1 is the human AT (HAT). The shorter isoform, designated MAT2 and RAT2, which contains an alternative N terminus, was formerly described in the rat as adrenal secretory serine protease (AsP) and has been shown to be involved in the processing of N-POMC-derived peptides. In contrast to the long isoform, neither MAT2 and RAT2 (AsP) contain a transmembrane domain nor a SEA domain but an N-terminal signal peptide to direct the enzyme to the secretory pathway. The C terminus, covering the catalytic triad, is identical in both isoforms. Immunohistochemically, MAT/RAT was predominantly expressed in tissues of the upper gastrointestinal and the respiratory tract—but also in the adrenal gland. Moreover, isoform-specific RT-PCR and quantitative PCR analysis revealed a complex expression pattern of the two isoforms with differences between mice and rats. These findings indicate a multifunctional role of these proteases beyond adrenal proliferation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
SERINE PROTEASES of the trypsin family are involved in many diverse physiological processes such as food digestion, blood coagulation, activation of complement, fertilization, defense against pathogens, cell signaling and development (1, 2) and have well-recognized roles in the pathology of diseases such as cancer (3). Recently, a serine protease in the rat adrenal gland was described, which is up-regulated during compensatory adrenal growth in the contralateral gland after unilateral adrenalectomy (4, 5, 6). It consists of a 28-kDa protein containing a secretory signal sequence together with the classical His/Asp/Ser catalytic triad and was, therefore, named the adrenal secretory serine protease (AsP). Based on the concept of Lowry and co-workers (7, 8, 9, 10) that the N-terminal proopiomelanocortin (N-POMC) regulates adrenal proliferation, AsP has been proposed to play a key role in this regulation process by specifically cleaving an N-terminal fragment of POMC (1–74 N-POMC) to generate a smaller peptide acting as an adrenal mitogen. Accordingly, in a recent study, we demonstrated the mitogenic activity of 1–28 POMC in vitro (11). In keeping with this concept, AsP is expressed in the outer rat adrenal cortex, the site of cell proliferation and was also found in a mouse adrenocortical cancer cell line (Y1 cells). Expression of antisense AsP RNA in Y1 cells reduced their growth rate, suggesting that AsP may also play a crucial role in neoplastic adrenal growth (4). Although an exhaustive survey has not been performed, AsP expression appeared to be limited to adrenal cells.

Comparison of the AsP sequence with that of other members of the trypsin family revealed high homology to the HAT [human airway trypsin-like protease (AT)]. However, HAT is considerably larger than AsP and contains an N-terminal transmembrane domain not shared by AsP. As the HAT gene does not appear to encode for the unique N-terminal portion of AsP, it was concluded that AsP is encoded by a different gene.

To further clarify this issue, we have characterized the gene encoding AsP in mouse and rat and have analyzed the expression profile of the gene products. The results demonstrate that the respective genes in rat and mouse code for both, a short secretory protease as well as a long isoform with a transmembrane and a SEA (63-kDa sea urchin sperm protein, enteropeptidase, agrin) domain. The long isoform is, indeed, the rodent homolog of HAT.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Adult male CD-1 mice and Wistar rats were purchased from Charles River Breeding Laboratories (Sulzfeld, Germany). Animals were kept in standard laboratory conditions with a 12-h light, 12-h dark cycle. All efforts were made to minimize both the suffering and number of animals used and all procedures were performed according to the accepted standards of animal care.

Isolation of RNA from solid tissues and blood
Total RNA was isolated by means of a commercially available modification (TRIzol; Invitrogen, Karlsruhe, Germany) of the one-step phenol/guanidinium thiocyanate method (12). Polyadenylated (PolyA+) RNA was isolated using the Oligotex mRNA Mini Kit (QIAGEN, Hilden, Germany). For the isolation of polyA+ RNA from total blood, we used the mRNA Isolation Kit for Blood/Bone Marrow (Roche, Mannheim, Germany). RNA amounts were determined by measuring the OD260 and quality was checked on agarose gels containing formaldehyde.

Rapid amplification of 5'- and 3'-cDNA ends (RACE)
RACE was performed using the simple modular architecture research tool (SMART) RACE cDNA Amplification Kit (CLONTECH, Alameda, CA) following the manufacturer’s instructions. PolyA+ RNA (0.5 µg) was used as starting material. The synthetic oligonucleotides used as primers for RACE-PCRs are depicted in Table 1Go. RACE products were analyzed in 1.5% agarose gels. Isolated bands were cut out of the gel, agarose was removed using Ultrafree-DA spin columns (Millipore, Eschborn, Germany). The cDNAs were introduced into the PCR-TOPO vector (Invitrogen, Karlsruhe, Germany) following the manufacturer’s instructions. Sequencing was performed by TOPLAB (Martinsried, Germany).


View this table:
[in this window]
[in a new window]
  		TABLE 1. Primers used in this study

 
RT-PCR
The reactions were carried out using total RNA (250 ng) as template and the QIAGEN OneStep RT-PCR Kit (QIAGEN) according to the manufacturer’s instructions. The synthetic oligonucleotides used as primers for RT-PCR are depicted in Table 1Go. RT-PCR was performed as follows, using a Mastercycler gradient (Eppendorf, Hamburg, Germany): 30 min of 50 C (room temperature), 15 min of 95 C inactivation of the reverse transcriptase and activation of Taq polymerase, followed by 40 cycles of 94 C for 1 min, 60 C for 1 min and 72 C for 2 min. The PCR products were analyzed in 1.5% agarose gels stained with ethidiumbromide.

Quantitative analysis of mouse AT (MAT) and rat AT (RAT) mRNA expression [quantitative PCR (QPCR)]
Total RNA was isolated form various tissues using TRI reagent (Sigma, Deisenhofen, Germany). Contaminating genomic DNA was removed by deoxyribonuclease I treatment and the RNA was repurified using TRI reagent. Two and half micrograms of the RNA were used to synthesize single-stranded cDNA using the ReverseIT kit (ABgene, Epsom, UK) in a 20-µl reaction using random hexamers as primer. The resulting cDNA was diluted to 100 µl and 1 µl used in each subsequent PCR. Specific Taqman probes and primer sets were designed using Primer Express (Applied Biosytems, Foster City, CA). The sequences of the primers and probes used were as follows: MAT1: Forward (5' CCA ATG CTA TCA CCG TCA AGA TT 3'); Reverse (5' GCC AGG AGC CCT ACC GTT AT 3'); Probe (5' FAM TTC ACT CCC TTT GCA GTA GCT TTC GTT GTC TAMRA 3'). MAT2: Forward (5' CTG AGA GCC AGG TGA AAT GAT TT 3'); Reverse (5' TGG TCA GTG AGT GCT ATC ATT AAG AA 3'); Probe (5' FAM CAG CTT CTG TTT TGT TGA TTT TGT TCT CAC CTT TT TAMRA 3'). RAT1: Forward (5' TCC TCC TTT GGC TTC AGG TAC A 3'); Reverse (5' TGT CCG TCC TAC TGC TTT GAG A 3'); Probe (5' FAM AGA ACA CGA AAG AGT CAT TCA GTT GAG AGG GA TAMRA 3'). RAT2: Forward (5' CAT GAA ATC AGG GTG AAA TGA GTT 3'); Reverse (5' CAG TGA GTG CTA GCG TTA AGA AAG A 3'); Probe (5' FAM CAG CTT CTG TTT TGT TGA TCT TCT TCT CGT GC TAMRA 3'). All primers and probes were synthesized by Sigma-Genosys (Pampisford, UK). Reactions were set up in triplicate in a total of 25 µl using QPCR ROX master mix (ABgene) following the manufacturer’s instructions and cycled in an ABI PRISM 7700 Sequence Detector under the following conditions: initial denaturation/activation of polymerase at 95 C for 15 min and then 40 cycles of 95 C for 15 sec and 60 C for 1 min. Expression levels of each variant were determined by comparison to standard curves generated using a 10-fold serial dilution of synthetic template containing the sequence of the complete amplicon. All results were normalized to expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The level of GAPDH expression in each tissue was assessed using a commercial primer/probe set following the manufacturer’s instructions (Applied Biosytems). Relative expression levels were calculated by comparison to a standard curve.

Immunohistochemistry
We used the recently described polyclonal rabbit antibody raised against the peptide DQDTENVLTQECGARPDLITLSEER corresponding to residues 23–47 of the full-length translated product of AsP (4). This peptide is part of both protease isoforms found in this study and is 100% conserved between mouse and rat. Therefore, this antibody recognizes both protease isoforms in both, mouse and rat. Longitudinal cryosections (10 µm) of mouse and rat embryos and mouse tongue and esophagus were blocked at room temperature for 2 h with 3% normal goat serum in 0.5x PAT (1x NaCl/Pi, 1% albumin, 0.5% Triton X-100) and then incubated overnight at 4 C with anti-AsP IgG (10 µg·µl-1 in 0.5x PAT). After three washes with NaCl/Pi, the sections were incubated for 2 h at room temperature with a Cy2 (cyanine 2-OSu bisfunctional)-conjugated affinity-purified goat antirabbit IgG (1:50; Rockland, Gilbertsville, PA) in 0.5x PAT. After being thoroughly washed, the sections were analyzed under a fluorescent microscope (Leica, Solms, Germany) and photographed with a Pixera charge-coupled device camera. The specificity of the MAT immunoreaction was verified by omitting the primary antibody and by using the peptide described above, as blocking peptide. We incubated 100 µg of peptide with MAT antibody (1:500) in a total volume of 500 µl for 2 h at room temperature before addition to the section.

Nucleotide and amino acid sequence analysis
Genome searches were performed using basic local alignment and search tool (BLAST) (13) at the National Center for Biotechnology Information databases (http://www.ncbi.nlm.nih.gov/), sequence similarities were studied using ClustalW (14) at European Molecular Biology Laboratory (EMBL) (http://www2.ebi.ac.uk/clustalw/), analysis of the modular domain architecture of proteins was performed with SMART (15) at EMBL (http://smart.embl-heidelberg.de/).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Isolation and nucleic acid sequence analysis of MAT and RAT cDNA clones
We used the published sequence of the rat AsP cDNA to perform a nucleotide blast (BLASTN) sequence analysis using the public domain GenBank sequence database. A mouse enhanced sequence tag (EST) clone, isolated from lung tissue, (dbEST BF143606) and genomic sequences of the mouse, very similar to the rat AsP, were identified. The EST clone was completely sequenced (Medigenomix, Martinsried, Germany). It encodes the mouse homolog of the HAT and shows a strong homology to rat AsP only in the C-terminal region. We named this protein MAT1. Based on the hypothesis that the AT gene of the mouse encodes a long, HAT-like and a shorter, AsP-like, isoform, we used RNA from the trachea and the adrenal gland of mouse and rat for the following experiments. RACE analysis was performed to obtain 5' and 3' ends of the cDNAs with primers depicted in Table 1Go. The sequences which we obtained were used to develop primers to amplify the full-length cDNAs of two different isoforms. A short isoform was amplified from adrenal- and a longer isoform from trachea RNA from both mouse and rat, respectively. We named the shorter protease isoform of the mouse MAT2, the longer isoform of the rat RAT1, and the shorter isoform of the rat, which is identical with the previously described AsP, RAT2. The sequences of the full-length cDNA clones were published in GenBank. Accession numbers are as follows: MAT1, AF448809; MAT2, AF539752; RAT1, AF453776.

Organization of the MAT gene
Comparison of the MAT1 and MAT2 cDNAs with the mouse genomic sequence using a BLAST homology search against the GenBank revealed that the MAT gene is located on the long arm of chromosome 5 at position E1 (GenBank accession no. NT_039307.1). It is composed of 10 exons and 9 introns, and spans about 70 kb (Fig. 1Go). The GT-AG rule for exon-intron boundaries is conserved. Interestingly, two similar genes—differentially expressed in squamous cell carcinoma (DESC) 1 and DESC2—encoding other trypsin-like proteases of the mouse—are also found on the long arm of chromosome 5 at position E1. The DESC genes have the same exon/intron organization as the AT gene (data not shown), suggesting that these genes are the result of a gene duplication.



View larger version (76K):
[in this window]
[in a new window]
  		FIG. 1. Structures of gene and deduced amino acid sequences, of MAT. Comparison of the nucleotide sequence of the MAT cDNA and the genomic sequence of chromosome 5E1 revealed the structure of the AT gene. The 10 exons are boxed, and the intervening sequences are not shown except for the exon/intron boundaries. Exon 6 contains an internal splicing site, which is used when the long isoform MAT1 is transcribed (lower box only). In case of the short isoform (MAT2), the complete exon 6 is used as the first exon (both boxes). The exon/intron boundary consensus sequence (GT/AG) is double underlined, the poly(A) signal is in bold and single underlined. Amino acids of the long isoform are numbered starting from the putative first initiating Met in exon 1; the amino acids of the short isoform are numbered with italic ciphers starting from the first putative initiating Met in exon 6. The essential triad is indicated by black triangles, the putative activation site is indicated with a white triangle, the SEA module and the transmembrane domain are indicated with gray and black shaded letters, respectively. The cysteine residues 173 and 291, which establish a conserved disulfide bond, linking the pro- and the catalytic domain, are circled. The disulfide bond is indicated by a dotted line.

 
Structural characterization of the Mus musculus AT isoforms
The full-length MAT1 cDNA sequence is 2043 nucleotides in length (built of exons 1–10) and comprises an open reading frame of 417 aa with a putative initiation codon at position 36. An A at position -3 from the start ATG fulfils the general Kozak rule for efficient translation (16, 17). The aa sequence shows strong identity (66%) with the HAT and the human DESC1 (37%) (Fig. 2Go).



View larger version (75K):
[in this window]
[in a new window]
  		FIG. 2. A, Derived amino acid alignment of MAT1, RAT1, and HAT with the human SEA domain containing serine protease DESC1. Completely conserved amino acids marked with * are in white letters; dashes represent gaps. Amino acids that are part of the catalytic triad are marked with a black arrow, the amino acids flanking the catalytic serine at position -6, 15, 16, 17, and 28, which determine the specificity of the substrate binding pocket are marked with white arrows. The predicted transmembrane regions are boxed. GenBank accession numbers of the sequences used: RAT, AAL50817; MAT, AAL47139; HAT, O60235; hDESC1, Q9UL52. B, Overall identity (%) of the aligned proteases.

 
MAT1 is a type II transmembrane serine protease (13) and possesses four structural regions (Fig. 3Go). The hydropathy plot (Fig. 3AGo) shows an N-terminal cytoplasmic region of 20 aa. It is followed by a hydrophobic transmembrane domain of 20 aa and a 145 aa stem region containing a SEA module (18). The serine protease domain of 232 aa with strong conservation of the amino acids surrounding the catalytic triad (His/Asp/Ser) is located at the C terminus (Fig. 3BGo). A putative cleavage site for proteolytic processing required to generate a mature form (Fig. 1Go) is tentatively assigned between Arg185 and Ile186, which is a highly conserved activation motif of serine proteases. A disulfide bridge linking the SEA module with the catalytic domain seems to be formed between Cys172 and Cys291, which are also conserved between type II transmembrane serine proteases (TTSPs) (19). MAT1 is likely to remain membrane bound after activation.



View larger version (29K):
[in this window]
[in a new window]
  		FIG. 3. A, Hydropathy plot of the deduced amino acid sequence of MAT1 and MAT2. The method of Hopp and Woods (52 ) was used (http://us.expasy.org/cgi-bin/protscale.pl). Hydrophobic residues show negative values, whereas hydrophilic residues show positive values. B, Schematic illustration of the structural comparison of the MAT isoforms. The amino acid number is indicated below each domain; exon boundaries are indicated with black, vertical bars. Amino acids belonging to the essential catalytic triad (H, D, S) are marked with white triangles; a possible cleavage and activation site is marked with a black triangle. CT, Cytoplasmic tail; SEA, SEA module; SP, signal peptide; TM, transmembrane domain.

 
The full-length MAT2 cDNA consists of 1626 nucleotides (build of exons 6–10 with an alternative transcriptional start point) and comprises an open reading frame of 279 aa (Fig. 1Go). The putative initiation codon is at position 88. The sequence surrounding the start ATG does not contain the Kozak consensus sequence.

MAT2 possesses three structural regions (Fig. 3Go). A hydrophobic signal peptide of 24 aa, which is supposed to direct the protein to the secretory pathway, is followed by a 23-aa linker peptide and the 232-aa catalytic domain. In this isoform, the cysteine residues forming the disulfide bridge, connecting the linker peptide with the catalytic domain, are in positions 34 and 153. The cleavage and activation site is between Arg47 and Ile48.

Both isoforms in all three AT-like proteases (MAT, RAT, HAT) share exons 7–10 and therefore have identical catalytic domains. The residues -6, 15, 16, 17, and 28 relative to the active serine are thought to determine the specificity of the substrate binding pocket in several serine proteases (20). All these positions are conserved in the ATs between the species examined, except position 15, where the human AT (HAT) features an Ile instead of Val (mouse, rat). The aspartic acid residue at position -6 from the catalytic serine is known to confer specificity for basic residues (20, 21).

Structural characterization of the Rattus norvegicus AT-like protease isoforms
The structural organization of the AT isoforms of the rat (R. norvegicus)—RAT1 and RAT2 (AsP)—is identical with the mouse protease isoforms described above.

Differential expression of MAT and RAT isoforms
RT- and QPCR data.
The expression pattern of the two isoforms was investigated by nonquantitative RT-PCR and by QPCR in various tissues of mice and rats. Using RT-PCR with primers specific for the long isoform, MAT1 was detected in the esophagus, lung, stomach, tongue, and trachea of mice (Fig. 4AGo, upper panel). There was also significant MAT1 mRNA expression in blood, brain, cerebellum, gut, heart, and spinal cord. These results were confirmed by QPCR (Fig. 4Go, A and B). Notably high amounts of MAT1 mRNA were detected in esophagus, tongue, and trachea, whereas small amounts were found in heart, lung, and adrenal gland. A similar expression pattern was found for MAT2. However, the relative abundance of MAT2 mRNA is distinctly lower than that of MAT1 expression (Fig. 4BGo)



View larger version (26K):
[in this window]
[in a new window]
  		FIG. 4. Specific distribution of MAT and RAT isoforms in various tissues of mouse and rat. A, RT-PCR analysis of the tissue distribution of MAT1/MAT2 in murine tissues. B, QPCR analysis of relative levels of MAT1/MAT2 mRNA in different tissues. C, RT-PCR analysis of the tissue distribution of RAT1/RAT2 in rat tissues. D, QPCR analysis of relative levels of RAT1/RAT2 mRNA in different tissues. All probes in RT-PCR were positive for ß-actin. As template, 500 ng of total RNA were used (exception in blood, when 50 ng polyA+ RNA served as template). Normalization in quantitative PCR was performed with GAPDH. ag, Adrenal gland; br, brain; cb, cerebellum; oe, esophagus; gt, gut; ht, heart; kd, kidney; ms, mussel; lg, lung; lv, liver; pg, pituitary gland; sc, spinal cord; st, stomach; tg, tongue; tr, trachea; y1, Y1 cells; -, no RNA.

 
The long AT isoform of the rat—RAT1—was found in the esophagus, tongue, and trachea using RT-PCR (Fig. 4CGo, upper panel). A faint band was obtained with RNA from the pituitary and no bands were found in adrenal gland, blood, brain, cerebellum, gut, heart, kidney, lung, liver, spinal cord, and stomach. QPCR analysis of MAT1 expression showed relatively high amounts of MAT1 mRNA in RNA derived from tongue, and lower levels in adrenal gland, pituitary gland, esophagus, lung, and trachea (Fig. 4DGo). In contrast, RAT2 (AsP) was mainly expressed in the adrenal gland as well as the tongue and showed also expression in the heart (Fig. 4Go, C and D). The relative amount of RAT2 mRNA in pituitary, esophagus, lung, and trachea was comparable to RAT1.

Immunohistochemistry.
Detailed immunohistochemical analysis of mouse embryonic and adult cryosections revealed many tissues to be positively stained (Fig. 5Go). Strong immunoreactivity was detected in adult tongue surface epithelium (Fig. 5Go, A and B) and the esophagus surface epithelium (Fig. 5Go, D and E). Staining of single cells was detected in adult trachea (Fig. 5FGo), embryonic lung (Fig. 5GGo), in the embryonic mouse gut (Fig. 5HGo). Weak staining was observed in the outer adrenal cortex region of adult mice (Fig. 5IGo). A similar pattern of immunoreactivity was detected in rat cryosections (data not shown). Preincubation of the antibody with the peptide used for immunization completely abolished immunoreactivity as shown in a tongue section in Fig. 5CGo.



View larger version (86K):
[in this window]
[in a new window]
  		FIG. 5. Immunohistochemical localization of MAT in various mouse tissues. The antibody used is raised against a sequence common to both isoforms of the protease. A, Cross section of the tongue of an adult mouse (100-fold magnification). Unstained papilla at the upper surface of the tongue are marked with white arrowheads. B, Detail of panel A, 400-fold magnification. C, Cross section of the tongue: the antibody was preincubated with a blocking peptide (400-fold magnification). D, Cross section of the esophagus of an adult mouse (400-fold magnification). E, Longitudinal section of the esophagus of an adult mouse (400-fold magnification), F, Longitudinal section of the trachea of an adult mouse (400-fold magnification). tc, Tracheal cartilages. G, Lung tissue of a mouse embryo, 23 d after fertilization (400-fold magnification). H, Cross section of the gut of a mouse embryo, 23 d after fertilization (400-fold magnification). I, Cross section of the adrenal gland of an adult mouse (200-fold magnification). zg, Zona glomerulosa; zf, zona fasciculata.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, the mouse and rat homologs of the HAT gene were characterized. Two different cDNAs were identified in both mouse and rat, encoding two different protease isoforms with significant structural differences and different expression patterns. One of these isoforms has been described previously in the rat as AsP (4).

We designated the long isoform MAT1 and RAT1, respectively, according to the human homolog HAT. These proteases exhibit the typical organization of TTSP: a short cytoplasmic tail at the N terminus followed by a transmembrane domain, a stem region, and the catalytic domain.

Whereas enteropeptidase, the first member of the family of TTSPs, was discovered nearly a century ago (22), only in recent years a rapidly growing number of TTSPs have been cloned including hepsin (23), HAT (24), corin (25), membrane-type serine protease 1 (MT-SP1) (26), matriptase (27), TMPRSS2 (28, 29), TMPRSS3 (30), seprase (31), TADG12 (32), TADG15 (33), Spinesin (34), DESC1 (35), and DESC2 and DESC3 (3). TTSPs differ in the structural organization of the modular stem region (19). In this region, MAT1/RAT1/HAT carry a so called SEA module. So far, only four other known members of the TTSP family carry a SEA module: enteropeptidase and DESC1, DESC2, and DESC3 (3, 36). The SEA module is an extracellular domain found in a variety of proteins (18). It was named after the first three proteins with an identified SEA module. All proteins containing a SEA module appear to be heavily glycosylated. In addition, the better characterized proteins all contain O-glycosidic-linked carbohydrates such as heparan sulfate that contribute considerably to their molecular masses. The common module might regulate or assist binding to neighboring carbohydrate moieties (18). On the other hand, the SEA module is a protein cleavage site of several transmembranous proteins (36). It was hypothesized that all membrane residing proteins containing a SEA module will undergo proteolytic cleavage and thereby generate ligand-receptor alliances (37). However, this may not be true in the case of MAT1 and RAT1 because the typical cleavage signal GSVVV, which is highly conserved in other SEA-containing proteins (38), is missing. Whereas enteropeptidase has a complex domain structure in the stem region [from the N terminus: SEA module, LDLa domain, CUB domain, MAM domain, CUB domain, LDLa domain, SR domain, trypsin-like serine protease domain (39)], DESC proteases have an identical structure as MAT1/RAT1/HAT because their stem regions carry only the SEA domain. As the murine DESC proteases are also located on the long arm of chromosome 5 and exhibit the same exon/intron organization it is, therefore, likely that these genes are the result of a gene duplication.

The shorter cDNA derived from the MAT/RAT gene by alternative transcription— MAT2/RAT2 (AsP)—encodes a secretory protease that was recently described as AsP (4, 5). The usage of alternative initial exons is a well-known phenomenon: in a recent study, a full-length mouse cDNA collection was compared with the draft of the mouse genome. It was shown that 31% of the genes with splice variation have alternative initial exons (40). However, only a few other reports have described genes of the TTSP family which encode both, a membrane spanning and a secretory isoform of a protease (41, 42).

MAT2 and RAT2 (AsP) contain a typical signal peptide, indicating that they are secretory proteins. However, it was shown that RAT2 (AsP) is retained on the surface of the cell after secretion (4, 40). It was suggested, that the high density of arginine residues on the posterior face of the active site anchors the enzyme to the cell surface. Also truncated human MT-SP1 lacking cytoplasmic and transmembrane domains remains bound to the cell surface of COS cells. It was suggested that this localization may be mediated via an interaction between MT-SP1 and another cell surface protein (40, 43). Interestingly, the HAT-like protease gene does not encode a short protease isoform. The unique sequence, encoding the N terminus of MAT2/RAT2 (AsP) has an equivalent in the human HAT gene. However, the insertion of 8 bp causes a frame shift and introduces a stop codon. All of our attempts to identify shorter HAT cDNAs in various human tissues by 5'-RACE PCR failed. Only the long isoform was demonstrated in cDNA from human trachea and adrenal gland (Hansen, I. A., unpublished results). Therefore, the existence of the short isoform is a true difference between man and mouse, and this raises the question whether the short isoform was lost during human evolution or represents a new development in rodents.

Our study indicates that the expression of MAT1/2 is more widespread than suggested from previous reports on the expression of HAT and AsP (4, 44). We used two methods to determine which tissues express which protease isoforms. The results of our RT- and QPCR experiments confirm each other for the most part with only two exceptions: in mouse esophagus and rat trachea, the short isoform was not detected by RT-PCR but found in the QPCR experiments (Fig. 4Go). In general, significant expression of the AT gene was observed in parts of the upper gastrointestinal tract (e.g. tongue, esophagus) and also in trachea and lung tissue with clear species differences between rat and mouse. Most notably, the short isoform [MAT2, RAT2 (AsP)] is highly expressed in the adrenal gland of the rat, whereas it is only expressed in relatively low levels in mouse tissue. The same is true for heart tissue. In a previous study, RAT2 (AsP) was also found in the pituitary gland of rats (45). Here we show that this is true for both isoforms in rat, whereas no AT expression could be detected in mouse tissue. Although MAT1/RAT1 is clearly expressed in rodent trachea suggesting a function similar to HAT, it is also found in the upper gastrointestinal tract and other tissues clearly indicating that its physiological role is not restricted to its production by ciliated cells of the bronchial epithelium.

The function of MAT1/RAT1 remains to be fully elucidated. The human homolog HAT has been isolated from mucoid sputum of patients with chronic airway disease (24). HAT has been reported to degrade fibrinogen, and it was suggested that it may play some role in the local host defense system of the upper airways (46). More recent data have shown that HAT may also activate protease-activated receptor 2 (PAR2) (47). PAR2 belongs to the family of protease activated G protein-coupled receptors (PAR1–4), which are activated by proteolytic exposure of an occult-tethered ligand (48, 49). PAR2 has been found to be activated by various proteases including trypsin, tryptase, and factor Xa. Moreover, PAR2 receptors are not only involved in airway physiology but have also been detected in a large number of other tissues including the adrenals, serving variable functions and potentially providing a target for HAT action outside the respiratory tract. In a recent study, it was demonstrated that RAT2 (AsP) specifically cleaves 1–74 N-POMC to generate a shorter adrenal mitogen and that inhibition of AsP expression reduces growth of mouse adrenal Y1 cells (4, 5). However, it is now clear that expression of the adrenal secretory serine protease (RAT2) is not restricted to the adrenal gland. This raises the question of a specific role of 1–74 N-POMC cleavage for mitogenesis in the adrenal gland because such cleavage may also occur in other tissues. Moreover, the expression of RAT2 (AsP) on the surfaces of esophagus and tongue indicates additional functions of the AsP beyond N-POMC cleavage.

Interestingly, melanocortin receptor expression has been found in exocrine glands, adrenal glands, and other peripheral organs of rodents (50, 51). In rat esophagus and adrenal glands, the expression pattern of MC5R overlaps with the ones we found for ATs. Thus, cleavage of N-terminal POMC peptides may occur in tissues other than adrenal gland, and this raises the question of which function these peptides have beyond the regulation of adrenal growth.

In summary, we have identified the gene encoding MAT, which encodes two different protease isoforms. Because of the specific tissue distribution of the enzyme and the complex expression pattern of its isoforms, it appears that MAT/RAT is a multifunctional protein, involved in various physiological processes. In particular, we provide evidence that the role of RAT2 (AsP) extends beyond adrenal mitogenesis.


   Acknowledgments
 
We acknowledge Geoffrey M. Attardo for language editing this manuscript.


   Footnotes
 
This work was supported by Deutsche Forschungsgemeinschaft (Al 203/7-3,4).

Abbreviations: aa, Amino acid, AsP, adrenal secretory serine protease; AT, airway trypsin-like protease; BLAST, basic local alignment and search tool; DESC, differentially expressed in squamous cell carcinoma; EST, enhanced sequence tag; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HAT, human airway trypsin-like protease; MAT, murine AT; MT-SP1, membrane-type serine protease 1; N-POMC, N-terminal POMC; PAR, protease-activated receptor; PolyA+, polyadenylated; POMC, proopiomelanocortin; QPCR, quantitative PCR; RACE, rapid amplification of cDNA ends; RAT, rat AT; SEA, 63-kDa sea urchin sperm protein, enteropeptidase, agrin; SMART, simple modular architecture research tool; TTSP, type II transmembrane serine protease.

Received July 24, 2003.

Accepted for publication December 17, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Barrett AJ, Rawlings ND, Woessner JF 1998 Handbook of proteolytic enzymes. 1st ed. Miami, FL: Academic Press
  2. Neurath H 1986 The versatility of proteolytic-enzymes. J Cell Biochem 32:35–49[CrossRef][Medline]
  3. Netzel-Arnett S, Hooper JD, Szabo R, Madison EL, Quigley JP, Bugge TH, Antalis AM 2003 Membrane anchored serine proteases: a rapidly expanding group of cell surface proteolytic enzymes with potential roles in cancer. Cancer Metastasis Rev 22:237–258[CrossRef][Medline]
  4. Bicknell AB, Lomthaisong K, Woods RJ, Hutchinson EG, Bennett HPJ, Gladwell RT, Lowry PJ 2001 Characterization of a serine protease that cleaves pro-{gamma}-melanotropin at the adrenal to stimulate growth. Cell 105:903–912[CrossRef][Medline]
  5. Bicknell AB 2002 Identification of the adrenal protease that cleaves pro-{gamma}-MSH: the dawning of a new era in adrenal physiology? J Endocrinol 172: 405–410
  6. Bicknell AB 2003 Identification of a serine protease involved with the regulation of adrenal growth. Ann NY Acad Sci 994:118–122[CrossRef][Medline]
  7. Estivariz FE, Iturriza F, Mclean C, Hope J, Lowry PJ 1982 Stimulation of adrenal mitogenesis by N-terminal proopiocortin peptides. Nature 297: 419–422
  8. Estivariz FE, Carino M, Lowry PJ, Jackson S 1988 Further evidence that N-terminal pro-opiomelanocortin peptides are involved in adrenal mitogenesis. J Endocrinol 116:201–206[Abstract/Free Full Text]
  9. Lowry PJ, Silas L, Mclean C, Linton EA, Estivariz FE 1983 Pro-{gamma}-melanocyte-stimulating hormone cleavage in adrenal-gland undergoing compensatory growth. Nature 306:70–73[CrossRef][Medline]
  10. Lowry PJ, Estivariz FE, Silas L, Linton EA, Mclean C, Crocombe K 1985 The case for pro-{gamma}-Msh as the adrenal growth-factor. Endocr Res 10:243–258
  11. Fassnacht M, Hahner S, Hansen IA, Kreutzberger T, Zink M, Adermann K, Jakob F, Troppmair J, Allolio B 2003 N-terminal proopiomelanocortin acts as a mitogen in adrenocortical tumor cells and decreases adrenal steroidogenesis. J Clin Endocrinal Metab 88:2171–2179[Abstract/Free Full Text]
  12. Chomczynski P, Sacchi N 1987 Single-step method of RNA isolation by acid guanidinium thiocyanate phenol chloroform extraction. Anal Biochem 162:156–159[Medline]
  13. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ 1990 Basic local alignment search tool. J Mol Biol 215:403–410[CrossRef][Medline]
  14. Thompson JD, Higgins DG, Gibson TJ 1994 Clustal-W—improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680[Abstract/Free Full Text]
  15. Schultz J, Copley RR, Doerks T, Ponting CP, Bork P 2000 SMART: a web-based tool for the study of genetically mobile domains. Nucleic Acids Res 28:231–234[Abstract/Free Full Text]
  16. Kozak M 1987 At least 6 nucleotides preceding the Aug initiator codon enhance translation in mammalian-cells. J Mol Biol 196:947–950[CrossRef][Medline]
  17. Kozak M 1999 Initiation of translation in prokaryotes and eukaryotes. Gene 234:187–208[CrossRef][Medline]
  18. Bork P, Patthy L 1995 The sea module—a new extracellular domain associated with O-glycosylation. Protein Sci 4:1421–1425[Medline]
  19. Hooper JD, Clements JA, Quigley JP, Antalis TM 2001 Type II transmembrane serine proteases—insights into an emerging class of cell surface proteolytic enzymes. J Biol Chem 276:857–860[Free Full Text]
  20. Kraut J 1977 Serine proteases—structure and mechanism of catalysis. Annu Rev Biochem 46:331–358[CrossRef][Medline]
  21. Ruhlmann A, Kukla D, Schwager P, Bartels K, Huber R 1973 Structure of the complex formed by bovine trypsin and bovine pancreatic trypsin inhibitor. Crystal structure determination and stereochemistry of the contact region. J Mol Biol 77:417–436[CrossRef][Medline]
  22. Lavallie ER, Rehemtulla A, Racie LA, Diblasio EA, Ferenz C, Grant KL, Light A, McCoy JM 1993 Cloning and functional expression of A cDNA-encoding the catalytic subunit of bovine enterokinase. J Biol Chem 268:23311–23317[Abstract/Free Full Text]
  23. Tsuji A, Torresrosado A, Arai T, Lebeau MM, Lemons RS, Chou SH, Kurachi K 1991 Hepsin, a cell membrane-associated protease—characterization, tissue distribution, and gene localization. J Biol Chem 266:16948–16953[Abstract/Free Full Text]
  24. Yamaoka K, Masuda K, Ogawa H, Takagi K, Umemoto N, Yasuoka S 1998 Cloning and characterization of the cDNA for human airway trypsin-like protease. J Biol Chem 273:11895–11901[Abstract/Free Full Text]
  25. Yan W, Sheng N, Seto M, Morser J, Wu QY 1999 Corin, a mosaic transmembrane serine protease encoded by a novel cDNA from human heart. J Biol Chem 274:14926–14935[Abstract/Free Full Text]
  26. Kim MG, Chen C, Lyu MS, Cho EG, Park D, Kozak C, Schwartz RH 1999 Cloning and chromosomal mapping of a gene isolated from thymic stromal cells encoding a new mouse type II membrane serine protease, epithin, containing four LDL receptor modules and two CUB domains. Immunogenetics 49:420–428[CrossRef][Medline]
  27. Lin CY, Anders J, Johnson M, Sang QXA, Dickson RB 1999 Molecular cloning of cDNA for matriptase, a matrix-degrading serine protease with trypsin-like activity. J Biol Chem 274:18231–18236[Abstract/Free Full Text]
  28. Jacquinet E, Rao NV, Rao GV, Hoidal JR 2000 Cloning, genomic organization, chromosomal assignment and expression of a novel mosaic serine proteinase: epitheliasin. FEBS Lett 468:93–100[CrossRef][Medline]
  29. Jacquinet E, Rao NV, Rao GV, Wang ZM, Albertine KH, Hoidal JR 2001 Cloning and characterization of the cDNA and gene for human epitheliasin. Eur J Biochem 268:2687–2699[Medline]
  30. Wallrapp C, Hahnel S, Muller-Pillasch F, Burghardt B, Iwamura T, Ruthenburger M, Lerch MM, Adler G, Gress TM 2000 A novel transmembrane serine protease (TMPRSS3) overexpressed in pancreatic cancer. Cancer Res 60:2602–2606[Abstract/Free Full Text]
  31. Goldstein LA, Ghersi G, PineiroSanchez ML, Salamone M, Yeh YY, Flessate D, Chen WT 1997 Molecular cloning of seprase: a serine integral membrane protease from human melanoma. Biochim Biophys Acta 1361:11–19[Medline]
  32. Underwood LJ, Tanimoto H, Wang YX, Shigemasa K, Parmley TH, O’Brien TJ 1999 Cloning of tumor-associated differentially expressed gene-14, a novel serine protease overexpressed by ovarian carcinoma. Cancer Res 59:4435–4439[Abstract/Free Full Text]
  33. Tanimoto H, Underwood LJ, Wang YX, Shigemasa K, Parmley TH, O’Brien TJ 2001 Ovarian tumor cells express a transmembrane serine protease: a potential candidate for early diagnosis and therapeutic intervention. Tumor Biol 22:104–114
  34. Yamaguchi N, Okui A, Yamada T, Nakazato H, Mitsui S 2002 Spinesin/TMPRSS5, a novel transmembrane serine protease, cloned from human spinal cord. J Biol Chem 277:6806–6812[Abstract/Free Full Text]
  35. Lang JC, Schuller DE 2001 Differential expression of a novel serine protease homologue in squamous cell carcinoma of the head and neck. Br J Cancer 84:237–243[CrossRef][Medline]
  36. Abe J, Fukuzawa T, Hirose S 2002 Cleavage of Ig-Hepta at a "SEA" module and at a conserved G protein-coupled receptor proteolytic site. J Biol Chem 277:23391–23398[Abstract/Free Full Text]
  37. Wreschner DH, McGuckin MA, Williams SJ, Baruch A, Yoeli M, Ziv R, Okun L, Zaretsky J, Smorodinsky N, Keydar I, Neophytou P, Stacey M, Lin HH, Gordon S 2002 Generation of ligand-receptor alliances by "SEA" module-mediated cleavage of membrane-associated mucin proteins. Protein Sci 11:698–706[CrossRef][Medline]
  38. Parry S, Silverman HS, McDermott K, Willis A, Hollingsworth MA, Harris A 2001 Identification of MUC1 proteolytic cleavage sites in vivo. Biochem Biophys Res Commun 283:715–720[CrossRef][Medline]
  39. Kitamoto Y, Yuan X, Wu QY, Mccourt DW, Sadler JE 1994 Enterokinase, the initiator of intestinal digestion, is a mosaic protease composed of a distinctive assortment of domains. Proc Natl Acad Sci USA 91:7588–7592[Abstract/Free Full Text]
  40. Zavolan M, van Nimwegen E, Gaasterland T 2002 Splice variation in mouse full-length cDNAs identified by mapping to the mouse genome. Genome Res 12:1377–1385[Abstract/Free Full Text]
  41. Hooper JD, Campagnolo L, Goodarzi G, Truong TN, Stuhlmann H, Quigley JP 2003 Mouse matriptase-2: identification, characterization and comparative mRNA expression analysis with mouse hepsin in adult and embryonic tissues. Biochem J 373:689–702[CrossRef][Medline]
  42. Kim DR, Sharmin S, Inoue M, Kido H 2001 Cloning and expression of novel mosaic serine proteases with and without a transmembrane domain from human lung. Biochim Biophys Acta 1518:204–209[Medline]
  43. Takeuchi T, Harris JL, Huang W, Yan KW, Coughlin SR, Craik CS 2000 Cellular localization of membrane-type serine protease 1 and identification of protease-activated receptor-2 and single-chain urokinase-type plasminogen activator as substrates. J Biol Chem 275:26333–26342[Abstract/Free Full Text]
  44. Takahashi M, Sano T, Yamaoka K, Kamimura T, Umemoto N, Nishitani H, Yasuoka S 2001 Localization of human airway trypsin-like protease in the airway: an immunohistochemical study. Histochem Cell Biol 115:181–187[Medline]
  45. Lowry, Lomthaisong K, Gladwell RT, Bicknell AB Pars intermedia pro-{gamma}-melanotropin and a cleft protease, a mitogenic conspiracy in the pituitary? Proc 192nd Meeting of the Society for Endocrinology, London, UK, 2001 (Abstract 2 OC18)
  46. Yoshinaga S, Nakahori Y, Yasuoka S 2003 Fibrinogenolytic activity of a novel trypsin-like enzyme found in human airway. J Med Invest 45:77–86
  47. Miki M, Nakamura Y, Takahashi A, Nakaya Y, Eguchi H, Masegi T, Yoneda K, Yasuoka S, Sone S 2003 Effect of human airway trypsin-like protease on intracellular free Ca2+ concentration in human bronchial epithelial cells. J Med Invest 50:95–107[Medline]
  48. Macfarlane SR, Seatter MJ, Kanke T, Hunter GD, Plevin R 2001 Proteinase-activated receptors. Pharmacol Rev 53:245–282[Abstract/Free Full Text]
  49. O’Brien PJ, Molino M, Kahn M, Brass LF 2001 Protease activated receptors: theme and variations. Oncogene 20:1570–1581[CrossRef][Medline]
  50. Labbe O, Desarnaud F, Eggerickx D, Vassart G, Parmentier M 1994 Molecular-cloning of a mouse melanocortin-5 receptor gene widely expressed in peripheral-tissues. Biochemistry 33:4543–4549[CrossRef][Medline]
  51. van der Kraan M, Adan RAH, Entwistle ML, Gispen WH, Burbach JPH, Tatro JB 1998 Expression of melanocortin-5 receptor in secretory epithelia supports a functional role in exocrine and endocrine glands. Endocrinology 139:2348–2355[Abstract/Free Full Text]
  52. Hopp TP, Woods KR 1983 A computer-program for predicting protein antigenic determinants. Mol Immunol 20:483–489[CrossRef][Medline]



This article has been cited by other articles:


Home page
 J Mol EndocrinolHome page
A. Hoeflich and M. Bielohuby
Mechanisms of adrenal gland growth: signal integration by extracellular signal regulated kinases1/2
J. Mol. Endocrinol., March 1, 2009; 42(3): 191 - 203.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Crit. Care Med.Home page
K. Shinagawa, J. A. Martin, V. A. Ploplis, and F. J. Castellino
Coagulation Factor Xa Modulates Airway Remodeling in a Murine Model of Asthma
Am. J. Respir. Crit. Care Med., January 15, 2007; 175(2): 136 - 143.
[Abstract] [Full Text] [PDF]

Home page
 J EndocrinolHome page
A. P Coll, M. Fassnacht, S. Klammer, S. Hahner, D. M Schulte, S. Piper, Y C L. Tung, B. G Challis, Y. Weinstein, B. Allolio, et al.
Peripheral administration of the N-terminal pro-opiomelanocortin fragment 1-28 to Pomc-/- mice reduces food intake and weight but does not affect adrenal growth or corticosterone production.
J. Endocrinol., August 1, 2006; 190(2): 515 - 525.
[Abstract] [Full Text] [PDF]

Home page
 Eur J EndocrinolHome page
S. Hahner, M. Fassnacht, F. Hammer, M. Schammann, D. Weismann, I. A. Hansen, and B. Allolio
Evidence against a role of human airway trypsin-like protease - the human analogue of the growth-promoting rat adrenal secretory protease - in adrenal tumourigenesis
Eur. J. Endocrinol., January 1, 2005; 152(1): 143 - 153.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Hansen, I. A.
Right arrow Articles by Allolio, B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hansen, I. A.
Right arrow Articles by Allolio, B.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals