Structure and activity of human pancreasin,a novel tryptic serine peptidase expressed primarily by the pancreas

In a search for genes encoding the serine peptidases prostasin and testisin, which are expressed mainly in prostate and testis, respectively, we identified a related, novel gene. Sequencing of cDNA allowed us to deduce the full amino acid sequence of the human gene product, which we term "pancreasin" because it is transcribed strongly in the pancreas. The idiosyncratic 6-exon organization of the gene is shared by a small group of tryptic proteases, including prostasin, testisin, and gamma-tryptase. Like the other genes, the pancreasin gene resides on chromosome 16p. Pancreasin cDNA predicts a 290-residue, N-glycosylated, serine peptidase with a typical signal peptide, a 12-residue activation peptide cleaved by tryptic hydrolysis, and a 256-amino acid catalytic domain. Unlike prostasin and other close relatives, human pancreasin and a nearly identical chimpanzee homologue lack a carboxyl-terminal membrane anchor, although this is present in 328-residue mouse pancreasin, the cDNA of which we also cloned and sequenced. In marked contrast to prostasin, which is 43% identical in the catalytic domain, human pancreasin is transcribed strongly in pancreas (and in the pancreatic ductal adenocarcinoma line, HPAC) but weakly or not at all in kidney and prostate. Antibodies raised against pancreasin detect cytoplasmic expression in HPAC cells. Recombinant, epitope-tagged pancreasin expressed in Chinese hamster ovary cells is glycosylated and secreted as an active tryptic peptidase. Pancreasin's preferences for hydrolysis of extended peptide substrates feature a strong preference for P1 Arg and differ from those of trypsin. Pancreasin is inhibited by benzamidine and leupeptin but resists several classic inhibitors of trypsin. Thus, pancreasin is a secreted, tryptic serine protease of the pancreas with novel physical and enzymatic properties. These studies provide a rationale for exploring the natural targets and roles of this enzyme.     

A novel isoform of a kallikrein-like protease, TLSP/hippostasin, (PRSS20), is expressed in the human brain and prostate

cDNAs encoding two splicing variants of a serine protease, termed hippostasin, were isolated by a PCR-based cloning strategy. The difference of 5' nucleotide sequence resulted in the variation in the amino terminal ends of the two, brain and prostate, types of human hippostasin. The longest ORF of the brain-type was 250 amino acids with a putative signal peptide, while that of the prostate-type was 282 amino acids. Homology search using the amino acid sequence revealed that prostate-type hippostasin was identical to TLSP (PRSS20), which is expressed in human primary keratinocytes (1). Transient expression analysis showed that both brain- and prostate-type TLSP/hippostasin were secreted into the conditioned medium as about 40 kDa proteins. Human TLSP/hippostasin showed 47% and 45% identity to trypsinogen II and kallikrein, respectively. In fact, the recombinant human TLSP/hippostasin efficiently cleaved Bz-Phe-Arg-4-methylcoumaryl-7-amide, a kallikrein substrate, and weakly cleaved other substrates for kallikrein and trypsin. Northern blot analysis detected a 1.3 kb band in the whole brain and a 1.4 kb band in the prostate and the lung. In situ hybridization revealed that it was expressed preferentially by the pyramidal neurons in the human hippocampus and secretory epithelial cells in the prostate. These results indicated that TLSP/hippostasin is involved in the functions of the human central nervous system and prostate and that it is a multifunctional protease present in various organs.     

A secretory protease inhibitor requires androgens for its expression in male sex accessory tissues but is expressed constitutively in pancreas.

A full length cDNA clone encoding a mouse prostatic secretory glycoprotein (p12) whose synthesis is dependent upon testicular androgens has been cloned and characterized. The predicted amino acid sequence of p12 shares extensive homology with several members of the Kazal family of secretory protease inhibitors, in particular the pancreatic secretory trypsin inhibitors. In agreement with sequence data, prostatic secretory p12, purified from mouse ventral prostate secretion, exhibits anti-trypsin activity. Steady-state levels of protease inhibitor mRNA in ventral prostate are reduced from approximately 0.06% in normal mice to undetectable after androgen withdrawal but are inducible within 4 h by re-administration of testosterone. Androgen-dependent expression of the secretory protease inhibitor mRNA was also observed in coagulating gland and seminal vesicle. In seminal vesicle, a tissue of different embryonic origin to the prostate, the kinetics of secretory protease inhibitor mRNA loss after castration are not as rapid as in the ventral prostate and coagulating gland. Low-level androgen independent expression was also observed in the pancreas. There appears to be a single gene for this secretory protease inhibitor and yet expression is markedly stimulated by testosterone in the sex accessory tissues and unaffected by this hormone in the pancreas.     

A novel protease homolog differentially expressed in breast and ovarian cancer.

BACKGROUND: Using differential display (DD), we discovered a new member of the serine protease family of protein-cleaving enzymes, named protease M. The gene is most closely related by sequence to the kallikreins, to prostate-specific antigen (PSA), and to trypsin. The diagnostic use of PSA in prostate cancer suggested that a related molecule might be a predictor for breast or ovarian cancer. This, in turn, led to studies designed to characterize the protein and to screen for its expression in cancer. MATERIALS AND METHODS: The isolation of protease M by DD, the cloning and sequencing of the cDNA, and the comparison of the predicted protein structure with related proteins are described, as are methods to produce recombinant proteins and polyclonal antibody preparations. Protease M expression was examined in mammary, prostate, and ovarian cancer, as well as normal, cells and tissues. Stable transfectants expressing the protease M gene were produced in mammary carcinoma cells. RESULTS: Protease M was localized by fluorescent in situ hybridization analysis to chromosome 19q13.3, in a region to which other kallikreins and PSA also map. The gene is expressed in the primary mammary carcinoma lines tested but not in the corresponding cell lines of metastatic origin. It is strongly expressed in ovarian cancer tissues and cell lines. The enzyme activity could not be established, because of difficulties in producing sufficient recombinant protein, a common problem with proteases. Transfectants were selected that overexpress the mRNA, but the protein levels remained very low. CONCLUSIONS: Protease M expression (mRNA) may be a useful marker in the detection of primary mammary carcinomas, as well as primary ovarian cancers. Other medical applications are also likely, based on sequence relatedness to trypsin and PSA.     

Mouse glandular kallikrein genes. Nucleotide sequence of cloned cDNA coding for a member of the kallikrein arginyl esteropeptidase group of serine proteases

A library of cloned cDNA to male mouse submaxillary gland poly(A)-containing RNA was constructed in the plasmid pBR322. Inserts containing sequences estimated to be in the 1-5% abundance class were identified by hybridization to radiolabeled cDNA and examined by nucleotide sequence analysis. A sequence coding for a peptide with 57% homology to the only complete kallikrein sequence reported to date (from pig pancreas) was identified by a computer search program. This insert appears to code for the COOH-terminal 149 amino acids of a protein presumed therefore to be a serine protease. Comparison of the predicted amino acid sequence of this protein with analogous sequences in the three characterized members of the mouse submaxillary gland kallikrein arginyl esteropeptidase group of enzymes revealed extensive homology, although not complete identity. Thus, there are at least four members of this enzyme family expressed in the mouse submaxillary gland.     

T-SP1: a novel serine protease-like protein predominantly expressed in testis

Here, we describe a novel member in the group of membrane-anchored chymotrypsin (S1)-like serine proteases, namely testis serine protease 1 (T-SP1), as it is principally expressed in testis tissue. The human T-SP1 gene encompasses 28.7 kb on the short arm of chromosome 8 and consists of seven exons. Rapid amplification of cDNA ends (RACE) experiments revealed that due to alternative splicing three different variants (T-SP1/1, -2, -3) are detectable in testis tissue displaying pronounced heterogeneity at their 3'-end. T-SP1/1 consists of an 18 amino acid signal peptide and of a 49 amino acid propeptide. The following domain with the catalytic triad of His(108), Asp(156), and Ser(250) shares sequence identities of 42% and 40% with the blood coagulation factor XI and plasma kallikrein, respectively. Only T-SP1/1 contains a hydrophobic part at the C-terminus, which provides the basis for cell membrane anchoring. Using a newly generated polyclonal anti-T-SP1 antibody, expression of the T-SP1 protein was found in the Leydig and Sertoli cells of the testis and in the epithelial cells of the ductuli efferentes. Notably, T-SP1 protein was also detectable in prostate cancer and in some ovarian cancer tissues, indicating tumor-related synthesis of T-SP1 beyond testis tissue.     

Protease II from Escherichia coli: sequencing and expression of the enzyme gene and characterization of the expressed enzyme.

Protease II gene of Escherichia coli HB101 was cloned and expressed in E. coli JM83. The transformant harboring a hybrid plasmid, pPROII-12, with a 2.4 kbp fragment showed 90-fold higher enzyme activity than the host. The whole nucleotide sequence of the inserted fragment of plasmid pPROII-12 was clarified by the dideoxy chain-terminating method. The sequence that encoded the mature enzyme protein was found to start at an ATG codon, as judged by comparison with amino terminal protein sequencing. The molecular weight of the enzyme was estimated to be 81,858 from the nucleotide sequence. The reactive serine residue of protease II was identified as Ser-532 with tritium DFP. The sequence around the serine residue is coincident with the common sequence of Gly-X-Ser-X-Gly, which has been found in the active site of serine proteases. Except for this region, protease II showed no significant sequence homology with E. coli serine proteases, protease IV and protease La (lon gene), or other known families of serine proteases. However, 25.3% homology was observed between protease II and prolyl endopeptidase from porcine brain. Although the substrate specificities of these two enzymes are quite different, it seems possible to classify protease II as a member of the prolyl endopeptidase family from the structural point of view.     

Human polyserase-2, a novel enzyme with three tandem serine protease domains in a single polypeptide chain

We have cloned a human cDNA encoding a new serine protease that has been called polyserase-2 (polyserine protease-2) because it is the second identified human enzyme with several tandem serine protease domains in its amino acid sequence. The first serine protease domain contains all characteristic features of these enzymes, whereas the second and third domains lack one residue of the catalytic triad of serine proteases and are predicted to be catalytically inactive. This complex domain organization is also present in the sequences of mouse and rat polyserase-2 and resembles that of polyserase-1, which also contains three serine protease domains in its amino acid sequence. However, polyserase-2 lacks additional domains present in polyserase-1, including a type II transmembrane motif and a low-density lipoprotein receptor A module. Enzymatic analysis demonstrated that both full-length polyserase-2 and its first serine protease domain hydrolyzed synthetic peptides used for assaying serine proteases. Nevertheless, the activity of the isolated domain was greater than that of the entire protein, suggesting that the two catalytically inactive serine protease domains of polyserase-2 may modulate the activity of the first domain. Northern blot analysis showed that polyserase-2 is expressed in fetal kidney; adult skeletal muscle, liver, placenta, prostate, and heart; and tumor cell lines derived from lung and colon adenocarcinomas. Finally, analysis of post-translational processing mechanisms of polyserase-2 revealed that, contrary to those affecting to the membrane-bound polyserase-1, this novel polyprotein is a secreted enzyme whose three protease domains remain as an integral part of a single polypeptide chain.     

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.     

苜蓿夜蛾丝氨酸蛋白酶基因cDNA序列的克隆与原核表达研究

【目的】丝氨酸蛋白酶(Serine protease,SP)是以丝氨酸为活性中心的重要的蛋白水解酶。在昆虫中,丝氨酸蛋白酶参与消化、发育、先天免疫反应和组织重建等重要的生理过程。本试验以苜蓿夜蛾Heliothis viriplaca为材料,克隆其丝氨酸蛋白酶基因的cDNA序列,再对该基因进行原核表达并对表达产物进行活性测定研究。【方法】从苜蓿夜蛾中肠中提取总RNA,通过RT-PCR和RACE技术,扩增获得丝氨酸蛋白酶基因cDNA全长序列,用大肠杆菌E.coli表达系统进行表达;再对表达的重组蛋白进行变性、纯化与复性,并以BTEE为底物进行活性测定。【结果】克隆得到的苜蓿夜蛾中肠丝氨酸蛋白酶基因命名为Hv SP,该基因已登录Gen Bank,登录号为KT907053。该基因全长1 017 bp,开放阅读框为886 bp,编码295个氨基酸,分子量约为30.8 ku,等电点为8.27,推导的氨基酸序列与其他昆虫丝氨酸蛋白酶氨基酸序列相似性在46%~92%之间。在Tris-HCl缓冲液中,p H为8.5时,复性的重组蛋白活性最高,为28.7 U/m L。荧光定量PCR结果表明,Hv SP基因的m RNA在苜蓿夜蛾的多个组织中特异性表达,且在中肠中表达量最高,但在唾腺中未检测到Hv SP的m RNA表达。【结论】该研究克隆了一个新的苜蓿夜蛾丝氨酸蛋白酶基因的cDNA序列,且原核表达后的重组蛋白经过变性、纯化及复性后具有活性,为进一步探索丝氨酸蛋白酶在昆虫体内的生理生化功能奠定了基础。     

Squamous cell carcinoma antigen is a new member of the serine protease inhibitors

We have cloned cDNA of squamous cell carcinoma antigen. Sequence analysis of the complete 1711 basepairs SCC antigen cDNA revealed that it coded 390 amino acids and no typical signal sequence in the NH2-terminus. Northern blot analysis of human squamous cell poly(A)+ RNA using this cDNA insert as the probe showed a single RNA species of about 1.7 kilobases. The cDNA was expressed in Escherichia coli and the product was detected by immunological methods using antibodies against SCC antigen, indicating that this cDNA encodes the entire SCC antigen sequence. The amino acid homology search revealed that SCC antigen was a member of the serine protease inhibitors family.     

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 of the cDNA for the serine protease homolog CAP37/azurocidin, a microbicidal and chemotactic protein from human granulocytes

Human cationic antimicrobial protein (CAP37) is a neutrophil granule protein with monocyte chemotactic and antibacterial activity. A CAP37 cDNA clone of 899 bp was isolated from an HL-60 cDNA library using degenerate oligonucleotide probes based on partial N-terminal sequence of the CAP37 protein. The cDNA sequence predicts an open reading frame of 753 bp encoding a protein of 251 amino acids. A 26-residue eukaryotic signal peptide and a potential 7 amino acid pro-peptide are present at the N-terminus of the protein. The cDNA sequence also predicts three N-linked glycosylation attachment sites and eight intramolecular cysteines. The deduced amino acid sequence of CAP37 shows 44, 42, and 32% homology at the amino acid level to neutrophil elastase, myeloblastin, and cathepsin G, respectively, suggesting that CAP37 is a member of the serine protease gene family. CAP37 does not possess serine protease activity probably due to mutations in two of three residues in the catalytic triad of the "charge relay system." Whereas CAP37 is expressed in undifferentiated HL-60 cells no message is detected in mature neutrophils.     

Identification of genes specifically expressed in the accumulated visceral adipose tissue of OLETF rats

The Otsuka Long-Evans Tokushima fatty (OLETF) rat is an animal model of type 2 diabetes, characterized by abdominal obesity, insulin resistance, hypertension, and dyslipidemia. To elucidate the underlying molecular mechanism of obesity and its related complications, we used representational difference analysis and identified the genes more abundantly and specifically expressed in the visceral adipose tissue (VAT) of obese OLETF rats compared with the diabetes-resistant counterpart, that is, Long-Evans Tokushima Otsuka (LETO) rats. By Northern blot analysis, we confirmed the differential expression of 13 genes, including 3 novel genes. The upregulated expression of well-characterized lipid metabolic enzymes, such as lipoprotein lipase, phosphoenolpyruvate carboxykinase, and cholesterol esterase, were observed in VAT of OLETF rats. We demonstrated the differential expression of secreted proteins in VAT of OLETF rats, such as thrombospondin 1 and contrapsin-like protease inhibitor. In contrast to lipid enzymes, the secreted proteins revealed exclusive mRNA expression and they were not detected in VAT of LETO rats. Furthermore, the novel genes OL-16 and OL-64 were also expressed specifically in VAT of OLETF rats and were absent in that of LETO rats and other tissues, including subdermal and brown adipose tissues. The C-terminal partial amino acid sequence of OL-64 revealed that it showed approximately 40% homology with alpha(1)-antitrypsin and it seemed to be a new member of the serine proteinase inhibitor (SERPIN) gene family. VAT of OLEFT rats had a unique gene expression profile, and the accumulated VAT-specific known and novel secreted proteins may play a role(s) in the pathogenesis of obesity and its related complications.     

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.     

Evaluation of MUC6 mucin tandem repeats

The MUC6 mucin was originally isolated from stomach mucus and is one of the major secreted mucins of the digestive tract. A full-length cDNA has not been isolated for this large molecule (greater than 15 kb) and it remains poorly studied. To circumvent the lack of reagents for investigating MUC6, we isolated a cDNA clone from a human fetal pancreatic duct cDNA library that encodes 282 amino acids of the MUC6 tandem repeat. A blast search with the sequence of this cDNA clone showed 90% homology with the original MUC6 (L07517) derived from a human stomach cDNA library and 95% homology both with AK096772, a MUC6-related protein isolated from a human prostate cDNA library and the human genome project clone AC083984. The MUC6 partial cDNA clone isolated from fetal pancreas was inserted into an epitope-tagged MUC1 mucin molecule in place of the native tandem repeat. This chimeric mucin was expressed in human pancreatic (Panc1) and colon (Caco2) carcinoma cell lines and purified for analysis of O-glycosylation by fast atom bombardment mass spectrometry (FAB-MS). The FAB-MS spectra showed O-glycans that had been detected previously on chimeric mucins carrying different tandem repeats, though the spectra for MUC1F/6TR mucins expressed in the Panc1 and Caco2 cells were very different. There was a paucity of O-glycosylation in Panc1 cells in comparison to Caco2 cells where many more structures were evident, and the most abundant glycans in Panc1 cells were sialylated.