Molecular cloning of cDNA for matriptase, a matrix-degrading serine protease with trypsin-like activity.

A major protease from human breast cancer cells was previously detected by gelatin zymography and proposed to play a role in breast cancer invasion and metastasis. To structurally characterize the enzyme, we isolated a cDNA encoding the protease. Analysis of the cDNA reveals three sequence motifs: a carboxyl-terminal region with similarity to the trypsin-like serine proteases, four tandem cysteine-rich repeats homologous to the low density lipoprotein receptor, and two copies of tandem repeats originally found in the complement subcomponents C1r and C1s. By comparison with other serine proteases, the active-site triad was identified as His-484, Asp-539, and Ser-633. The protease contains a characteristic Arg-Val-Val-Gly-Gly motif that may serve as a proteolytic activation site. The bottom of the substrate specificity pocket was identified to be Asp-627 by comparison with other trypsin-like serine proteases. In addition, this protease exhibits trypsin-like activity as defined by cleavage of synthetic substrates with Arg or Lys as the P1 site. Thus, the protease is a mosaic protein with broad spectrum cleavage activity and two potential regulatory modules. Given its ability to degrade extracellular matrix and its trypsin-like activity, the name matriptase is proposed for the protease.     

Vaccinia virus encodes two proteins that are structurally related to members of the plasma serine protease inhibitor superfamily.

Nucleotide sequencing adjacent to the right inverted terminal repetition of the vaccinia virus genome revealed two genes encoding polypeptides that are structurally related to members of the plasma serine protease inhibitor superfamily (SPI). Inclusion in the superfamily is based on extensive amino acid sequence similarities as well as a consensus sequence adjacent to the active-site region near the carboxyl ends of the proteins. The genes designated SPI-1 and SPI-2 are located 10,000 and 17,000 base pairs from the right end of the genome, respectively. The predicted SPI-1 polypeptide is 11 amino acids longer than that of SPI-2, and the deduced masses are 40,471 and 38,125 daltons, respectively. Similarities between SPI-1 and SPI-2 are indicated by the percentage of identical amino acids (44%) and corresponding hydrophobicity plots. The maximum amino acid sequence diversity occurs precisely in the putative active-site region, suggesting that SPI-1 and SPI-2 may inhibit different proteases. SPI-2 is homologous to a previously described cowpox virus gene (D. J. Pickup, B. S. Ink, W. Hu, C. A. Ray, and W. K. Joklik, Proc. Natl. Acad. Sci. USA 83:7698-7702, 1986). Evidence for a cowpox virus homolog of SPI-1 was obtained by DNA hybridization. Thus, the presence of two genes that belong to the plasma serine protease inhibitor superfamily may be characteristic of orthopoxviruses.     

Adipsin, the adipocyte serine protease: gene structure and control of expression by tumor necrosis factor.

We have isolated, mapped and sequenced adipsin, the adipocyte differentiation-dependent serine protease gene. This gene, which is present in a single form in the mouse, spans 1.7 kilobases and contains five exons. While the basic exon structure characteristic of serine protease genes is conserved in adipsin, there is also a fusion of two exons that are separate in other serine proteases. The sequence data also suggests a mechanism of alternative splicing which appears to account for the generation of two adipsin mRNA species differing by only three nucleotides and encoding two different signal peptides. To investigate the control of adipsin expression we have examined the effects of tumor necrosis factor (TNF) on adipocytes. The level of adipsin RNA is dramatically decreased by hormone treatment, but the change occurs more slowly than for other fat cell mRNAs, such as glycerophosphate dehydrogenase. These results show that adipsin is a novel serine protease gene whose expression is regulated by a macrophage-derived factor which modulates expression of other adipocyte-specific RNAs.     

cDNA cloning of porcine brain prolyl endopeptidase and identification of the active-site seryl residue

Prolyl endopeptidase is a cytoplasmic serine protease. The enzyme was purified from porcine kidney, and oligonucleotides based on peptide sequences from this protein were used to isolate a cDNA clone from a porcine brain library. This clone contained the complete coding sequence of prolyl endopeptidase and encoded a polypeptide with a molecular mass of 80,751 Da. The deduced amino acid sequence of prolyl endopeptidase showed no sequence homology with other known serine proteases. [3H]Diisopropyl fluorophosphate was used to identify the active-site serine of prolyl endopeptidase. One labeled peptide was isolated and sequenced. The sequence surrounding the active-site serine was Asn-Gly-Gly-Ser-Asn-Gly-Gly. This sequence is different from the active-site sequences of other known serine proteases. This difference and the lack of overall homology with the known families of serine proteases suggest that prolyl endopeptidase represents a new type of serine protease.     

Complete amino acid sequence of streptokinase and its homology with serine proteases

The complete amino acid sequence of streptokinase has been determined by automated Edman degradation of its cyanogen bromide and proteolytic fragments. The protein consists of 415 amino acid residues. Sequence microheterogeneity was found at two positions. The NH2-terminal 245 residues of streptokinase are homologous to the sequences of several serine proteases including bovine trypsin and Streptomyces griseus proteases A and B. The sequence alignment suggests that the active-site histidine-57 has changed to a glycine in streptokinase. The other active-site residues, aspartyl-102 and serine-195, are, however, present at the expected positions. Streptokinase also contains internal sequence homology between the NH2-terminal 173 residues and a COOH-terminal 162-residue region between residues 254 and 415. Moderate homology in predicted secondary structures also exists between these two regions. Although streptokinase is not a protease, these observations suggest that it has evolved from a serine protease by gene duplication and fusion. A COOH-terminal region of about 80 residues is apparently deleted from the second half of the duplicated structures. These observations further suggest that the three-dimensional structure of streptokinase likely contains two independently folded domains, each homologous to serine proteases.     

Cloning and expression of novel mosaic serine proteases with and without a transmembrane domain from human lung

Two cDNAs encoding novel mosaic proteins with a serine protease domain and potential regulatory modules, consisting of a protein kinase substrate and a low-density lipoprotein receptor, were cloned from a human lung cDNA library by PCR. One with a transmembrane domain (MSPL) and the other without one (MSPS) comprise 581 and 537 amino acids, respectively. Except for the C-terminal ends, the two isoforms had an identical serine protease domain exhibiting 42, 39 and 43% identity with those of plasma kallikrein, hepsin and transmembrane protease serine 2, respectively. Both genes were predominantly expressed in human lung, placenta, pancreas and prostate.     

Nucleotide sequence of the gene for aqualysin I (a thermophilic alkaline serine protease) of Thermus aquaticus YT-1 and characteristics of the deduced primary structure of the enzyme

Aqualysin I is an alkaline serine protease which is secreted into the culture medium by Thermus aquaticus YT-1, an extreme thermophile [Matsuzawa, H., Hamaoki, M. & Ohta, T. (1983) Agric. Biol. Chem. 47, 25-28]. The gene encoding aqualysin I was cloned into Escherichia coli using synthetic oligodeoxyribonucleotides as hybridization probes. The nucleotide sequence of the cloned DNA was determined. The primary structure of aqualysin I, deduced from the nucleotide sequence, agreed with the NH2-terminal sequence previously reported and the determined amino acid sequences, including the COOH-terminal sequence, of the tryptic peptides derived from aqualysin I. Aqualysin I comprised 281 amino acid residues and its molecular mass was determined to be 28,350. On alignment of the whole amino acid sequence, aqualysin I showed high sequence homology with the subtilisin-type serine proteases, and 43% identity with proteinase K, 37-39% with subtilisins and 34% with thermitase. Extremely high sequence identity was observed in the regions containing the active-site residues, corresponding to Asp32, His64 and Ser221 of subtilisin BPN'. The nucleotide sequence of the cloned DNA (1105 nucleotides) revealed that it contains the entire gene encoding aqualysin I and one open reading frame without a translational stop codon. Therefore, aqualysin I was considered to be produced as a large precursor, which contains a NH2-terminal portion, the protease and a COOH-terminal portion. The G + C content of the coding region for aqualysin I was 64.6%, which is lower than those of other Thermus genes (68-74%). The codon usage in the aqualysin I gene was rather random in comparison with that in other Thermus genes.     

Polymorphism and evolution of collagenolytic serine protease genes in crustaceans.

Two genomic DNA fragments encoding crustacean collagenolytic serine protease genes show coding fragments that span 1522-1526 base pairs and contain seven exons encoding the complete amino acid sequence of two enzymes, CHYA and CHYB. As in serine protease genes from other organisms, the region coding for the residues around the active site is split by two introns. Although the introns differ from those of other organisms in size and nucleotide sequence, their number and location are more or less the same as found in mammalian chymotrypsin or elastase genes that evolved lately, but different for trypsin genes. Meanwhile, the junction that occurs between the propeptide and the maturation site is only found in the shrimp genes. This is also the case for the junction located 13 amino acids after the active site aspartic acid in these genes. Between 40 and 50 copies of the genes are reported by Southern analysis. Seven different genes within ChyA Pv family present 0-6% base changes, whereas five different genes belonging to ChyB Pv family show changes of up to 27% in the short studied portion of exon 4. This last family presents a mosaic organization of the coding parts, which are also expressed in the hepatopancreas of the shrimp as the variant PVC5 cDNA.     

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.     

Synthesis of peptidyl methylcoumarin esters as substrates and active-site titrants for the prohormone processing proteases Kex2 and PC2

Peptidyl methylcoumarin amides are well established as model substrates for understanding protease specificity, but the corresponding methylcoumarin esters have attracted scant attention despite their potential utility in active-site titration and mechanistic characterization. We have devised techniques for the synthesis and deprotection of extended peptidyl methylcoumarin esters in good to moderate yields, and we have demonstrated their suitability for steady-state characterization and active-site titration of the Saccharomyces cerevisiae processing protease Kex2. Additionally, we have used one of these compounds to active-site titrate the homologous enzyme PC2, which had not previously been feasible using other types of substrates. These compounds should thus prove widely suitable for use as substrates and active-site titrants not only for proteases of the prohormone processing family but also for a wide range of other serine proteases.     

Characterization of a prolyl endopeptidase from Flavobacterium meningosepticum. Complete sequence and localization of the active-site serine.

A prolyl endopeptidase was purified from Flavobacterium meningosepticum. It was digested with trypsin. Two oligonucleotides, based on tryptic peptide sequences and used in PCR experiments, amplified a 300-base pair (bp) fragment. A 2.4-kilobase EcoRI fragment that hybridized to the 300-bp probe was cloned in lambda ZAP and sequenced from both strands. It contains a reading frame of 2115 bp, encoding the complete protein sequence of 705 amino acids. Ion-spray mass spectrometry experiments demonstrated the presence of an NH2-terminal signal peptide: the periplasmic mature protease is 685 residues in length for a molecular mass of 76784 Da. The prolyl endopeptidase showed no general sequence homology with known protein sequences except with that of porcine brain prolyl endopeptidase. In order to identify the active-site serine, the prolyl endopeptidase was labeled with [3H]diisopropyl fluorophosphate. One labeled peptide was purified and sequenced. The active-site serine was located in position 536 within the sequence GRSNGG. This sequence is different from the active-site sequence of the trypsin (GDSGGP) and subtilisin (GTSMAS) families.     

“两广二号”家蚕抗病毒基因Bmlipase-1和BmSP-2克隆与原核表达

本研究以优良杂交品种"两广二号"家蚕为试材,克隆了该杂交品种家蚕两个抗家蚕核型多角体病毒(BmNPV)基因:脂肪酶基因Bmlipase-1和丝氨酸蛋白酶基因BmSP-2,测序并分别与不同品种蚕的同源基因序列进行比较。结果显示,"两广二号"家蚕Bmlipase-1基因ORF长度为885bp,编码294个氨基酸,BmSP-2扩增长度为855bp,编码284个氨基酸;它们的核苷酸和推导氨基酸序列同源性皆达92%以上,Bmlipase-1更保守,同源性大于99%";两广二号"家蚕的Bmlipase-1基因脂肪酶活化部位和BmSP-2基因酶催化三联体位点的氨基酸残基与不同品种蚕的完全相同。以上结果说明这两个抗病毒基因在蚕的遗传进化过程中高度保守,提示其可能在机体消化或者免疫防御方面起着重要生理作用。将这两个抗病毒基因在大肠杆菌BL21中进行融合表达,获得的融合Bmlipase-1和BmSP-2蛋白分子量分别为47kD和42kD左右。     

Amino acid sequence of human D of the alternative complement pathway

The primary structure of human D, the serine protease activating the C3 convertase of the alternative complement pathway, has been deduced by sequencing peptides derived from various chemical (CNBr and o-iodosobenzoic acid) and enzymatic (trypsin, lysine protease, Staphylococcus aureus V8 protease, and chymotrypsin) cleavages. Carboxypeptidase A was also used to confirm the COOH-terminal sequence. The peptides were purified by high-pressure liquid chromatography. The proposed sequence of human D contains 222 amino acids and has a calculated molecular weight of 23 748. It exhibits a high degree of homology with other serine proteases, especially around the NH2-terminus as well as the three residues corresponding to the active-site His-57, Asp-102, and Ser-195 (chymotrypsinogen numbering). This sequence homology is highest (40%) with plasmin, intermediate (35%) with pancreatic serine proteases, such as elastase, trypsin, chymotrypsin, and kallikrein, and least (30%) with the serum enzymes thrombin and factor X. D, however, exhibits only minimal amino acid homology with the other sequenced complement serine proteases, Clr (25%) and Bb (20%). The substitution of a basic lysine for a neutral amino acid three residues NH2-terminal to the active-site serine as well as a small serine residue for a bulky aromatic amino acid at position 215 (chymotrypsinogen numbering) in the binding pocket may be important in determining the exquisite substrate specificity of D. The presence of His-40 which interacts with Asp-194 (chymotrypsinogen numbering) to stabilize other serine protease zymogens [Freer, S. T., Kraut, J., Robertus, J. D., Wright, H. T., & Xuong, N. H. (1970) Biochemistry 9, 1997] argues in favor of such a D precursor molecule.     

Structure of the Mature Streptococcal Cysteine Protease Exotoxin mSpeB in Its Active Dimeric Form

Invasive infections of Streptococcus pyogenes are dependent on the cysteine protease streptococcal pyrogenic exotoxin B. Previous structures of the enzyme have not disclosed the proper active-site configuration. Here, the crystal structure of the mature enzyme is presented to 1.55 Å, disclosing a homodimer. A serine from one subunit inserts into the active site of the other to donate to the oxyanion hole and coordinates the ligand proximal to the active-site cysteine. Dimerization is unique to the mature form and is clearly a prerequisite for catalysis. The present structure supports a tripartite switch system that is triggered upon dimerization and substrate binding: (1) liberation of the active-site histidine from an inactive configuration, (2) relocation of residues blocking the substrate binding pockets and (3) repositioning of two active-site tryptophans to settle in the active configuration. Based on the present structure, the active site of clan CA cysteine proteases is expanded and a detailed mechanism of the deacylation mechanism is proposed. The results may have applications for the development of protease inhibitors specific to bacterial cysteine proteases.     

Deletion analysis of the essentiality of penicillin-binding proteins 1A, 2B and 2X of Streptococcus pneumoniae

Abstract An internal fragment from each of the penicillinebinding protein (PBP) 1A, 2B and 2X genes of Streptococcus pneumoniae , which included the region encoding the active-site serine residue, was replaced by a fragment encoding spectinomycin resistance. The resulting constructs were tested for their ability to transform S. pneumoniae strain R6 to spectinomycin resistance. Spectinomycin-resistant transformants could not be obtained using either the inactivated PBP 2X or 2B genes, suggesting that deletion of either of these genes was a lethal event, but they were readily obtained using the inactivated PBP 1A gene. Analysis using the polymerase chain reaction confirmed that the latter transformants had replaced their chromosomal copy of the PBP 1A gene with the inactivated copy of the gene. Deletion of the PBP 1A gene was therefore tolerated under laboratory conditions and appeared to have little effect on growth or susceptibility to benzylpenicillin.     

Complement genes C1r and C1s feature an intronless serine protease domain closely related to haptoglobin

The exon-intron structure of the human complement C1s gene displays a striking similarity with that of the gene encoding haptoglobin, a peculiar transport protein distantly related to the serine proteases. While the protease regions of the serine zymogens are typically encoded by multiple exons, the protease domains of C1s and of its genetically linked and functionally interacting homolog C1r are encoded as intronless domains, not unlike a region of haptoglobin, which in fact is devoid of proteolytic activity. The close similarity of the C1s gene with haptoglobin includes the precise conservation of exon-intron junctions and it extends to upstream exons encoding the short repeats typical of several complement components, but found also in other functionally unrelated proteins. Additional evidence of the common ancestry of C1r, C1s and haptoglobin is the presence, within the protease domain, of a set of sequence markers that distinguish these three proteins from all known serine proteases. The finding of vertebrate serine protease genes with an uninterrupted protease-encoding exon supports the definition of a novel evolutionary branch of this gene family and rules out the hypothesis that regards this unusual exon as an irrelevant byproduct of the extravagant functional divergence of haptoglobin.     

A mouse serine protease TESP5 is selectively included into lipid rafts of sperm membrane presumably as a glycosylphosphatidylinositol-anchored protein

We have previously indicated that at least in mouse, sperm serine protease(s) other than acrosin probably act on the limited proteolysis of egg zona pellucida to create a penetration pathway for motile sperm, although the participation of acrosin cannot be ruled out completely. A 42-kDa gelatin-hydrolyzing serine protease present in mouse sperm is a candidate enzyme involved in the sperm penetration of the zona pellucida. In this study, we have PCR-amplified an EST clone encoding a testicular serine protease, termed TESP5, and then screened a mouse genomic DNA library using the DNA fragment as a probe. The DNA sequence of the isolated genomic clones indicated that the TESP5 gene is identical to the genes coding for testicular testisin and eosinophilic esp-1. Immunochemical analysis using affinity-purified anti-TESP5 antibody revealed that 42- and 41-kDa forms of TESP5 with the isoelectric points of 5.0 to 5.5 are localized in the head, cytoplasmic droplet, and midpiece of cauda epididymal sperm probably as a membranous protein. Moreover, these two forms of TESP5 were selectively included into Triton X-100-insoluble microdomains, lipid rafts, of the sperm membranes. These results show the identity between TESP5/testisin/esp-1 and the 42-kDa sperm serine protease. When HEK293 cells were transformed by an expression plasmid carrying the entire protein-coding region of TESP5, the recombinant protein produced was released from the cell membrane by treatment with Bacillus cereus phosphatidylinositol-specific phospholipase C, indicating that TESP5 is glycosylphosphatidylinositol-anchored on the cell surface. Enzymatic properties of recombinant TESP5 was similar to but distinguished from those of rat acrosin and pancreatic trypsin by the substrate specificity and inhibitory effects of serine protease inhibitors.     

Chromosome localization of two human serine protease genes to region 14q11.2----q12 by in situ hybridization

Two human serine protease genes have been cloned. One corresponds to CTLA1, the human equivalent of the mouse cytotoxic cell protease gene Ctla-1, and the other is novel. Both genes were localized to 14q11.2----q12 by in situ hybridization. This result confirms the assignment of human CTLA1 to 14q11.2----q12 and provides new mapping data for another human serine protease gene located in the same chromosome region.