Dog mastocytoma tryptase: affinity purification, characterization, and amino-terminal sequence

A tryptic protease with the characteristics of a mast cell tryptase was purified from dog mastocytoma cells propagated in nude mice. Partial amino acid sequence of the mastocytoma tryptase revealed unexpected differences in comparison with other mast cell and leukocyte granule protease sequences. Extraction from mastocytoma homogenates at high ionic strength, followed by gel filtration and benzamidine affinity chromatography yielded a product with several closely spaced bands (Mr 30,000-32,000) on gel electrophoresis and a single N-terminal sequence. Nondenaturing analytical gel filtration revealed an apparent Mr of 132,000, suggesting noncovalent association as a tetramer. Studies with peptide p-nitroanilides indicated pronounced substrate preferences, with P1 arginine preferred to lysine. Benzoyl-L-Lys-Gly-Arg-p-nitroanilide was the best of the substrates screened. Inhibition by diisopropyl fluorophosphate and tosyllysine chloromethyl ketone indicated that the enzyme is a serine protease. Like the tryptases of human mast cells, mastocytoma tryptic protease was inhibited by NaCl, resistant to inactivation by alpha 1-proteinase inhibitor and plasma, and stabilized by heparin. Comparison of the N-terminal 24 residues of mastocytoma tryptase revealed 80% identity with the more limited sequence reported for human lung tryptase, and surprisingly, closer homology to serine proteases of digestion and clotting than to other leukocyte granule proteases sequenced to date, including mast cell chymase. The N-terminal isoleucine is the homolog of trypsinogen Ile-16 which becomes the new N-terminus upon cleavage of the activation peptide. Thus, the tryptase N-terminus is related to the catalytic domain of activated serine proteases, and lacks the N-terminal regulatory domains found in most clotting and complement serine proteases. These findings provide further evidence that tryptases are unique serine proteases and that they may be less closely related in evolution and function than are other leukocyte granule proteases described to date.     

Dog mast cell chymase: molecular cloning and characterization

We cloned and characterized a cDNA coding for the complete amino acid sequence of dog mast cell chymase. The cDNA was identified by screening a dog mastocytoma cDNA library with an oligonucleotide probe based on the amino acid sequence of a fragment of dog mastocytoma chymase. The deduced amino acid sequence reveals a putative 21-residue prepropeptide followed by a catalytic domain of 228 residues. The primary structure of the preproenzyme shares features with rat mucosal mast cell chymase (RMCP II), several lymphocyte-associated proteases, and neutrophil cathepsin G. The common characteristics include an apparent activation peptide terminating in glutamic acid, strict conservation of an octapeptide (residues 9-16) in the N-terminal portion of the catalytic domain, and the presence of only six cysteines available for intramolecular disulfide bond formation. However, dog chymase differs in being modified by N-glycosylation. Although the dog chymase catalytic domain exhibits a similar level of sequence identity when compared with both RMCP II and the rat connective tissue mast cell chymase RMCP I (58% and 61%, respectively), the dog enzyme most closely resembles RMCP I in its high predicted net charge (+16) and in the presence of serine at the base of its putative primary substrate binding pocket. The dog chymase differs strikingly from dog mast cell tryptase in the preprosequence and in the structure of the catalytic domain. Therefore, chymase appears not to be closely related to tryptase and may not share a mechanism of activation, even though both enzymes are packaged and released together.     

Cloning of the cDNA and gene of mouse mast cell protease-6. Transcription by progenitor mast cells and mast cells of the connective tissue subclass

The cDNA and gene for mouse mast cell protease-6 (MMCP-6) have been sequenced and show MMCP-6 to be translated as a prepro-enzyme with a 21-amino acid hydrophobic leader peptide, a 10-amino acid activation peptide, and a 245-amino acid mature enzyme. The mature form of the enzyme has 73% amino acid sequence identity with human and dog mast cell tryptases. The MMCP-6 gene includes 6 exons, with a total span of 1.8 kilobases. A 208-base pair intron was defined which separates the 5'-untranslated sequence of MMCP-6 from the translation initiation codon, thereby presenting a gene organization which distinguishes tryptic serine proteases from chymotryptic serine proteases of the mast cell secretory granule. By RNA blot analysis with a gene-specific probe, MMCP-6 has a unique subclass distribution in being transcribed in mouse connective tissue mast cells but undetectable in mucosal mast cells. MMCP-6 is the first serine protease of any class to be shown to be significantly transcribed in progenitor, bone marrow-derived mast cells, which can reconstitute both mucosal mast cell and connective tissue mast cell populations in mast cell-deficient mice.     

Rat mast cell protease 4 is a beta-chymase with unusually stringent substrate recognition profile

Activated mast cells release a variety of potent inflammatory mediators including histamine, cytokines, proteoglycans, and serine proteases. The serine proteases belong to either the chymase (chymotrypsin-like substrate specificity) or tryptase (trypsin-like specificity) family. In this report we have investigated the substrate specificity of a recently identified mast cell protease, rat mast cell protease-4 (rMCP-4). Based on structural homology, rMCP-4 is predicted to belong to the chymase family, although rMCP-4 has previously not been characterized at the protein level. rMCP-4 was expressed with an N-terminal His tag followed by an enterokinase site substituting for the native activation peptide. The enterokinase-cleaved fusion protein was labeled by diisopropyl fluorophosphate, demonstrating that it is an active serine protease. Moreover, rMCP-4 hydrolyzed MeO-Suc-Arg-Ala-Tyr-pNA, thus verifying that this protease belongs to the chymase family. rMCP-4 bound to heparin, and the enzymatic activity toward MeO-Suc-Arg-Ala-Tyr-pNA was strongly enhanced in the presence of heparin. Detailed analysis of the substrate specificity was performed using peptide phage display technique. After six rounds of amplification a consensus sequence, Leu-Val-Trp-Phe-Arg-Gly, was obtained. The corresponding peptide was synthesized, and rMCP-4 was shown to cleave only the Phe-Arg bond in this peptide. This demonstrates that rMCP-4 displays a striking preference for bulky/aromatic amino acid residues in both the P1 and P2 positions.     

A novel trypsin-like serine protease (hepsin) with a putative transmembrane domain expressed by human liver and hepatoma cells

Recombinant clones with cDNA inserts coding for a new serine protease (hepsin) have been isolated from cDNA libraries prepared from human liver and hepatoma cell line mRNA. The total length of the cDNA is approximately 1.8 kilobases and includes a 5' untranslated region, 1251 nucleotides coding for a protein of 417 amino acids, a 3' untranslated region, and a poly(A) tail. The amino acid sequence coded by the cDNA for hepsin shows a high degree of identity to pancreatic trypsin and other serine proteases present in plasma. It also exhibits features characteristic of zymogens to serine proteases in that it contains a cleavage site for protease activation and the highly conserved regions surrounding the His, Asp, and Ser residues that participate in enzyme catalysis. In addition, hepsin lacks a typical amino-terminal signal peptide. Hydropathy analysis of the protein sequence, however, revealed a very hydrophobic region of 27 amino acids starting 18 residues downstream from the apparent initiator Met. This region may serve as an internal signal sequence and a transmembrane domain. This putative transmembrane domain could be involved in anchoring hepsin to the cell membrane and orienting it in such a manner that its carboxyl terminus, containing the catalytic domain, is extracellular.     

cDNA cloning of granzyme C, a granule-associated serine protease of cytolytic T lymphocytes

A cDNA clone corresponding to the complete amino acid sequence of a putative protease CCP2 of murine cytotoxic T lymphocytes was isolated and sequenced. The clone encodes a 248-residue long serine esterase. The deduced N-terminal amino acid sequence is identical over 40 residues to that of granzyme C, a protease of unknown function present in granules of cytotoxic lymphocytes. Analysis of the sequence of granzyme C/CCP2 reveals high homology to other granzyme proteases, i.e. granzyme A (40%) and granzyme B (67%) and to rat mast cell protease II (46%). The amino acids lining the specificity pocket are well conserved between granzyme B, C, and rat mast cell protease II, but not granzyme A, suggesting a similar general specificity of these three proteases.     

Proteolytic susceptibility of the serine protease inhibitor trappin-2 (pre-elafin): evidence for tryptase-mediated generation of elafin

A number of serine, cysteine, metallo- and acid proteases were evaluated for their ability to proteolytically cleave the serine protease inhibitor trappin-2, also known as pre-elafin, and to release elafin from its precursor. None of the metalloproteases or acid proteases examined cleaved trappin-2, while serine and cysteine proteases preferentially cleaved trappin-2 within its non-inhibitory N-terminal moiety. Cathepsin L, cathepsin K, plasmin, trypsin and tryptase were able to release elafin by cleaving the Lys 38 -Ala 39 peptide bond in trappin-2. However, purified tryptase appeared to be efficient at releasing elafin. Incubation of trappin-2 with purified mast cells first challenged with anti-immunoglobulin E or calcium ionophore A23187 resulted in the rapid generation of elafin. This proteolytic release of elafin from trappin-2 was inhibited in the presence of a tryptase inhibitor, suggesting that this mast cell enzyme was involved in the process. Finally, ex vivo incubation of trappin-2 with sputum from cystic fibrosis patients indicated the production of a proteolytic immunoreactive fragment with the same mass as that of native elafin. This cleavage did not occur when preincubating the sputum with polyclonal antibodies directed against tryptase. Taken together, these findings indicate that tryptase could likely be involved in the maturation of trappin-2 into elafin under physiological conditions.     

Purification and molecular cloning of a novel serine protease from the centipede, Scolopendra subspinipes mutilans

A novel serine protease, named as scolonase, was purified and characterized from the tissue of the Korean centipede, Scolopendra subspinipes mutilans. Purified scolonase showed an apparent molecular weight of 25 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and an isoelectric point of 4.8 on isoelectric focusing gel. Scolonase was able to preferentially hydrolyze arginine over lysine at the cleavage site among the several synthetic peptide substrates. Scolonase has also a potent fibrinolytic activity by converting human Glu-plasminogen to activated plasmin due to the specific cleavage of the molecule at the peptide bond Arg(561)-Val(562). The enzyme activity of scolonase was completely inhibited by phenylmethanesulfonyl fluoride and difluorophosphate. The cDNA encoding scolonase was cloned from the cDNA library of the centipede constructed with oligonucleotide probe, which was designed on the basis of the N-terminal amino acid sequence of scolonase. The deduced complete amino acid sequence of scolonase demonstrated that the protein is composed of 277 amino acids including 33 amino acids as a leader sequence, and that it has significant sequence homology with other serine proteases.     

Molecular cloning of complementary DNA encoding one of the human pancreatic protease E isozymes

We have cloned a DNA from a human pancreatic cDNA library using a cloned rat pancreatic elastase 1 cDNA as a probe, and determined its nucleotide sequence. This cDNA contains a coding region of 810 nucleotides which encodes a 270-amino-acid protein. The deduced amino acid sequence shows less than 60% homologies with rat and porcine pancreatic elastase 1, although its substrate binding region is homologous with those of the above elastases 1. When this deduced amino acid sequence was compared with known amino acid sequences of pancreatic proteases other than elastases, it was found to contain an amino acid sequence which was highly homologous with the N-terminal amino acid sequence of porcine pancreatic protease E. We also purified human pancreatic protease E isozymes from human pancreatic juice, and determined their N-terminal amino acid sequences. One of the isozymes does not hydrolyze elastin but does hydrolyze a synthetic substrate. Endoglycosidase F digests glycoside bonds of the isozyme. These results suggest that the cDNA cloned by us corresponded to one of the human protease E isozymes.     

Amino acid sequence of a mouse mucosal mast cell protease

The amino acid sequence has been determined of a mouse mucosal mast cell protease isolated from the small intestines of mice infected with Trichinella spiralis. The active protease contains 226 residues. Those corresponding to the catalytic triad of the active site of mammalian serine proteases (His-57, Asp-102, and Ser-195 in chymotrypsin) occur in identical positions. A computer search for homology indicates 74.3% and 74.1% sequence identity of the mouse mast cell protease compared to those of rat mast cell proteases I and II (RMCP I and II), respectively. The six half-cystine residues in the mouse mast cell protease are located in the same positions as in the rat mast cell proteases, cathepsin G, and the lymphocyte proteases, suggesting that they all have identical disulfide bond arrangements. At physiological pH, the mouse and rat mucosal mast cell proteases have net charges of +3 and +4, respectively, as compared to +18 for the protease (RMCP I) from rat connective tissue mast cells. This observation is consistent with the difference in solubility between the mucosal and connective tissue mast cell proteases when the enzymes are extracted from their granules under physiological conditions.     

The primary structure of the 32-kDa subunit of human replication protein A

Replication protein A (RP-A) is a complex of three polypeptides of molecular mass 70, 32, and 14 kDa, which is absolutely required for simian virus 40 DNA replication in vitro. We have isolated a cDNA coding for the 32-kDa subunit of RP-A. An oligonucleotide probe was constructed based upon a tryptic peptide sequence derived from whole RP-A, and clones were isolated from a lambda gt11 library containing HeLa cDNA inserts. The amino acid sequence predicted from the cDNA contains the peptide sequence obtained from whole RP-A along with two sequences obtained from tryptic peptides derived from sodium dodecyl sulfate-polyacrylamide gel-purified 32-kDa subunit. The coding sequence predicts a protein of 29,228 daltons, in good agreement with the electrophoretically determined molecular mass of the 32-kDa subunit. No significant homology was found with any of the sequences in the GenBank data base. The protein predicted from the cDNA has an N-terminal region rich in glycine and serine along with two acidic and two basic segments. Monoclonal antibodies have been raised against the 70- and 32-kDa subunits of RP-A. The cloned cDNA has been overexpressed in bacteria using an inducible T7 expression system. The protein made in bacteria is recognized by a monoclonal antibody that is specific for the 32-kDa subunit of RP-A. This monoclonal antibody against the 32-kDa subunit inhibits DNA replication in vitro.     

Characterization of cDNA clones encoding mouse proteinase 3 (myeloblastine) and cathepsin G

 Serine proteases are the most abundant granule constituents of several major hematopoietic cell lineages. Due to their high abundance and their strict tissue specificity they have become important phenotypic cell markers used for studies of various aspects of hematopietic cell development. Using a polymerase chain reaction (PCR)-based strategy for the isolation of trypsin-related serine proteases, we were able to isolate cDNAs for two of the major neutrophil and monocyte serine proteases in the mouse, cathepsin G and mouse protease 3 (myeloblastin). The internal PCR fragments were used as probes to screen a mouse mast cell cDNA library and a cDNA library originating from a mouse monocytic cell line (WEHI-274.1). Full-length cDNAs for mouse cathepsin G and proteinase 3 were isolated and their complete sequences were determined. Northern blot analysis revealed expression of cathepsin G in immature cells of the monocyte macrophage lineage but also in the connective tissue mast cell line MTC. Proteinase 3 was expressed in several cell lines of myelo-monocytic origin and in one B-cell line, but not in any of the other cell lines tested. The isolation of cDNAs for mouse cathepsin G and mouse proteinase 3, together with the previous characterization of the gene for mouse N-elastase, and the entire or partial amino acid sequences for porcine azurocidine, equine N-elastase and proteinase 3, rat, dog, and rabbit cathepsin Gs in evolutionary relatively distantly related mammalian species, indicates that these four members of the serine protease family have been maintained for more than 100 million years of mammalian evolution. This latter finding indicates a strong evolutionary pressure to maintain specific immune functions associated with these neutrophil and monocyte proteases. All amino acid positions of major importance for the cleavage site selection have also been fully conserved between mouse and human proteinase 3 and a few minor changes have occurred between mouse and human cathepsin G. Received: 3 August 1996 / Revised: 24 February 1997     

Molecular cloning of complementary DNA for human medullasin: an inflammatory serine protease in bone marrow cells

Medullasin, an inflammatory serine protease in bone marrow cells, modifies the functions of natural killer cells, monocytes, and granulocytes. We have cloned a medullasin cDNA from a human acute promyelocytic cell (ML3) cDNA library using oligonucleotide probes synthesized from the information of N-terminal amino acid sequence of natural medullasin. The cDNA contained a long open reading frame encoding 237 amino acid residues beginning from the second amino acid of natural meduallasin. The deduced amino acid sequence of medullasin shows a typical serine protease structure, with 41% homology with pig elastase 1.     

Cloning and characterization of chymotrypsin- and trypsin-like cDNAs from the gut of the Hessian fly [Mayetiola destructor (Say)

Fifteen unique cDNA clones encoding trypsin- or chymotrypsin-like proteins were cloned and characterized from a gut cDNA library derived from Hessian fly [Mayetiola destructor (Say)] larvae. Based on sequence similarities, the cDNAs were sorted into five gene groups, which were named MDP1 to MDP5. Two of the gene groups, MDP1 and MDP2, encoded chymotrypsin-like proteins; the other three encoded putative trypsins. All deduced proteins have conserved His(87), Asp(136), and Ser(241) residues for the catalytic triad and three pairs of cysteine residues for disulfide bridge configurations. The substrate specificity determination residue at position 235 was also conserved in the putative trypsins and chymotrypsins. In addition, all the deduced protein precursors had a typical secretion signal peptide and activation peptide. Northern blot analysis revealed that all these gene groups were exclusively expressed in the larval stage. The expression profiles for each gene group differed significantly in different ages of the larva, as well as in different tissues. Protease activity analysis of gut extract, using specific inhibitors, demonstrated that serine proteases were the major digestive enzymes in the gut of M. destructor larvae. Serine protease inhibitors inhibited as much as 90% proteolytic activities of gut extract, whereas inhibitors specific to other proteases, including cysteine proteases, aspartic proteases, and metallo-proteases, inhibited only 10-24% of gut protease activity.     

Purification and cloning of a novel serine protease, RNK-Met-1, from the granules of a rat natural killer cell leukemia.

We have purified a 30-kDa serine protease (designated RNK-Met-1) from the granules of the rat large granular lymphocyte leukemia cell line (RNK-16) that hydrolytically cleaves model peptide substrates after methionine, leucine, and norleucine (Met-ase activity). Utilizing molecular sieve chromatography, heparin-agarose, chromatography, and reverse-phase high pressure liquid chromatography, RNK-Met-1 was purified to homogeneity and 25 NH2-terminal amino acids were sequenced. By using the polymerase chain reaction, oligonucleotide primers derived from amino acids at position 14-25 and from a downstream active site conserved in other serine protease genes were used to generate a 534-base pair cDNA clone encoding a novel serine protease from RNK-16 mRNA. This cDNA clone was used to isolate a full-length 867-base pair RNK-Met-1 cDNA from an RNK-16 lambda-gt11 library. The open reading frame predicts a mature protein of 238 amino acids with two potential sites for N-linked glycosylation. The cDNA also encodes a leader peptide of at least 20 amino acids. The characteristic Ile-Ile-Gly-Gly amino acids of the NH2 terminus and the His, Asp, and Ser residues that form the catalytic triad of serine proteases were both conserved. The amino acid sequence has less than 45% identity with any other member of the serine protease family, indicating that RNK-Met-1 is distinct and may itself represent a new subfamily of serine proteases. Northern blot analysis of total cellular RNA detected a single 0.9-kilobase mRNA in the in vitro and in vivo variants of RNK-16 and in spleen-derived plastic-adherent rat lymphokine-activated killer cells. RNK-Met-1 mRNA was not detectable in freshly isolated rat splenocytes, thymocytes, brain, colon, and liver or activated nonadherent rat splenocytes and thymocytes. These data indicate that RNK-Met-1 is a serine protease with unique activity that is expressed in the granules of large granular lymphocytes.     

Cloning of the cDNA and gene for mouse mast cell protease 4. Demonstration of its late transcription in mast cell subclasses and analysis of its homology to subclass-specific neutral proteases of the mouse and rat

Based on the amino-terminal amino acid sequence of the mature form of mouse mast cell protease 4 (MMCP-4), previously identified in peritoneal connective tissue mast cells (CTMC) and Kirsten sarcoma virus-immortalized mast cells (KiSV-MC), a 26-mer oligonucleotide probe was constructed and used to clone cDNAs for MMCP-4 from a KiSV-MC1 cDNA library. MMCP-4 is the first secretory granule serine protease of CTMC to be molecularly cloned. Using a cDNA probe derived from the 3'-untranslated portion of the MMCP-4 cDNA, the gene for MMCP-4 and a second highly related gene (mouse mast cell protease-like, MMCP-L) were cloned from a BALB/c mouse genomic DNA library and sequenced entirely, including approximately 2 kilobases of the 5'-flanking region. MMCP-4 and MMCP-L have five exons of identical length, four introns of nearly identical length, and approximately 900 base pairs of 5'-flanking DNA with sequence similarity by dot matrix analysis. By RNA blot analysis with gene-specific probes for MMCP-4 (bases 497-633 of the cDNA) and MMCP-L (bases 502-638 of the cDNA), mRNA for MMCP-4 was present in KiSV-MC5, CTMC, and the intestine of a mouse infected with the parasite Nippostrongylus brasiliensis markedly enriched for mucosal mast cells (MMC); MMCP-L mRNA was detected only in the intestine of the N. brasiliensis-infected mouse. MMCP-4 mRNA was not expressed in normal mouse intestine or in interleukin 3-dependent bone marrow-derived mast cells, which can serve as precursors to both MMC and CTMC. This finding suggests that MMCP-4 is transcribed relatively late in the development of both the CTMC and MMC subclasses and underscores the fact that mouse bone-marrow-derived mast cells are immature mast cells, rather than tissue culture equivalents of the MMC subclass.