Expression and excretion of human pancreatic secretory trypsin inhibitor in lipoprotein-deletion mutant of Escherichia coli

We constructed a gene coding for the 56-amino acid human pancreatic secretory trypsin inhibitor (PSTI), and ligated it on a plasmid downstream from the trp promoter and the signal peptide sequence of alkaline phosphatase. The resulting plasmid was transfected into a lipoprotein deletion mutant (Escherichia coli JE5505) and the plasmid-carrying cells were induced with 3-indoleacrylic acid. A considerable amount (50 micrograms/ml culture) of the mature PSTI protein was detected in the culture supernatant. The excreted PSTI was identical to the natural PSTI protein with respect to the trypsin-inhibiting activity, the N-terminal and the C-terminal amino acid sequences and the amino acid composition.     

Expression of human pancreatic secretory trypsin inhibitor in Saccharomyces cerevisiae

Human pancreatic secretory trypsin inhibitor (PSTI) cDNA was expressed in Saccharomyces cerevisiae using the yeast acid phosphatase PHO5 promoter. The product encoded by the PSTI-coding cDNA was correctly processed in yeast cells, and the PSTI molecules were efficiently secreted into the medium. The amino acid composition and the N-terminal amino acid sequence of the secreted PSTI molecules were identical to those of the authentic PSTI polypeptides from human pancreas, and the product exhibited trypsin-inhibitory activity.     

Production of specific monoclonal antibodies against the active sites of human pancreatic secretory trypsin inhibitor variants by in vitro immunization with synthetic peptides

Specific monoclonal antibodies against the active sites of two genetically engineered pancreatic secretory trypsin inhibitor (PSTI) variants (PSTI 0 and PSTI 4) were produced. The protease inhibitors PSTI 0 and PSTI 4 differ only by three amino acid substitution at their active sites. PSTI 0 inhibits trypsin, whereas PSTI 4 inhibits human granulocyte elastase and chymotrypsin. Immunization was performed in vitro with a synthetic heptapeptide that covers the mutated region of the protein. For this purpose in vitro culture conditions for the production of specific monoclonal antibodies against synthetic peptides were improved. The monoclonal antibodies obtained react specifically with the corresponding protease inhibitor variant. Competition experiments with trypsin and human elastase demonstrate that the protease displace the monoclonal antibody from the active site of PSTI 0 and PSTI 4 respectively.     

Molecular cloning and nucleotide sequence of human pancreatic secretory trypsin inhibitor (PSTI) cDNA

We have isolated and sequenced a cDNA clone coding for the human pancreatic secretory trypsin inhibitor (PSTI) from a human pancreatic cDNA library. The predicted product consists of 79 amino acids, and contains no apparent functional polypeptide other than PSTI. Southern blot analysis suggests that there is one copy of PSTI gene per haploid genome. This gene seems to be expressed not only in pancreas, but also in gastric mucosa, since a Northern blot analysis demonstrated the presence of a poly(A) RNA of the same size as in the pancreas. A comparison of sequences between human PSTI mRNA and mouse epidermal growth factor (EGF) mRNA revealed a high homology, suggesting that they share a common ancestral DNA sequence.     

Demonstration of pancreatic secretory trypsin inhibitor in serum-free culture medium conditioned by the human pancreatic carcinoma cell line CAPAN-1

Serum-free culture medium conditioned by an established human pancreatic adenocarcinoma cell line, CAPAN-1, contains copious amounts of immunoreactivity due to pancreatic secretory trypsin inhibitor (PSTI) as demonstrated by radioimmunoassay. The immunoreactive substance was purified from the conditioned medium to apparent homogeneity by trypsin affinity and gel filtration chromatography with an overall recovery of 40%, and its primary structure was determined by Edman degradation. The immunoreactive substance is a peptide of 56 amino acid residues with a calculated molecular weight of 6,241. Its amino acid composition, primary structure, and inhibitory effect against trypsin are indistinguishable from those of human pancreatic juice PSTI, indicating that this substance is PSTI itself. This is the first direct demonstration that tumor cells secrete PSTI in vitro. When CAPAN-1 was inoculated into a nude mouse, it produced a tumor and the tumor synthesized human PSTI in vivo, as demonstrated by the fact that the tumor extract contained 99.0 +/- 26.2 ng of human PSTI/mg of protein, while PSTI was not detected in extracts from other tissues examined. Furthermore, high levels of human PSTI (14.3 +/- 2.6 ng/ml) were detected in the serum of tumor-bearing mice but not in that of nontumor-bearing mice, suggesting that PSTI secreted from the tumor appears in the blood circulation. Taken together, these results strongly support the view that the serum levels of PSTI are elevated in cancer-bearing patients due to secretion of this peptide from tumor cells per se.     

Trypsin Inhibitor Polymorphism: Multigene Family Expression and Posttranslational Modification

Trypsin inhibitors from winter pea seeds (c.v. Frilene) have been purified and shown to consist of six protease inhibitors (PSTI I, II, III, IVa, IVb, and V). Based on amino acid composition, molecular mass, and N-terminal sequence, the six inhibitors are closely related to one another and belong to the Bowman–Birk family of inhibitors. To define the relations among them, molecular mass and amino acid composition of peptides obtained from digestion with trypsin were determined. The sequence and the biosynthetic mechanism of the isoform formation have been partially resolved for four major isoforms. Two isoinhibitor forms (PSTI IVa, IVb) in pea seeds are due to expression of two distinct genes; PSTI IVa has four amino acid replacements when its sequence is compared with the sequence of PSTI IVb. Two others (PSTI I, II) result from posttranslational proteolytic cleavage of nine C-terminal residues of forms PSTI IVa and IVb, respectively.     

Molecular cloning and nucleotide sequence of Streptomyces griseus trypsin gene.

Streptomyces griseus trypsin (E.C. 3.4.21.4) is one of the major extracellular proteinase, which is secreted by S. griseus. The gene encoding S. griseus trypsin was isolated from a S. griseus genomic library by using a synthetic oligonucleotide probe. Fragments containing the gene for S. griseus trypsin were characterized by hybridization and demonstration of proteolytic activity in S. lividans. Deduced amino acid sequence from the nucleotide sequence suggests that S. griseus trypsin is produced as a precursor, consisting of three portions; an amino-terminal pre sequence (32 amino acid residues), a pro sequence (4 residues), and the mature trypsin. The S. griseus trypsin consists of 223 amino acids with a computed molecular weight of 23,112. The existence of proline at the pro and mature junction suggests that the processing of S. griseus trypsin is non-autocatalytic.     

Expression in Escherichia coli and characterization of human growth-hormone-releasing factor

A chemically synthesized DNA sequence, coding for the 44 amino acid residues of human growth-hormone-releasing factor (GRF) preceded by a tryptophan codon, was cloned in frame with Escherichia coli trpE gene within a pBR322-derived plasmid. GRF was expressed in E. coli as a fused polypeptide chain (TrpE-GRF) and then the GRF amino acid sequence was released from the fused protein by specific chemical cleavage at the tryptophan residue using o-iodosobenzoic acid. The thioether group of the methionine residue of GRF was converted in the sulfonium salt derivative, in order to prevent irreversible oxidation of methionine to the sulfone derivative by the o-iodosobenzoic acid reagent. GRF was purified by HPLC and characterized in terms of amino acid composition after acid hydrolysis, protein sequencing and gel electrophoretic behaviour. These data clearly established that the biosynthetic GRF was identical to the natural one, except for the lack of amidation at the carboxyl-terminal amino acid. Far-ultraviolet circular dichroism measurements established that both biosynthetic and natural GRF are devoid of secondary structure in aqueous solution at neutral pH, whereas both peptide samples achieve a high percentage of helical structure in the presence of trifluoroethanol.     

Trypsin inhibitor from the seeds of Acacia confusa.

A trypsin inhibitor (ACTI) was isolated and purified from the seeds of Acacia confusa by gel filtration, and trypsin-Sepharose 4B column affinity chromatography. The molecular weight of ACTI was found to be 21,000 +/- 1,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and amino acid composition analysis. ACTI contained four half-cystine and no methionine residues, and was rich in aspartic acid, glutamic acid, glycine, leucine, and lysine residues. The native trypsin inhibitor was composed of two polypeptide chains, and it inhibited trypsin and alpha-chymotrypsin stoichiometrically at the molar ratio of 1:1 and 2:1, respectively. The amino-terminal sequence analysis of the A. confusa trypsin inhibitor A and B chains revealed a more extensive homology with Acacia elata and silk tree trypsin inhibitors, and a less extensive homology with Kunitz soybean trypsin inhibitor.     

Activities of herpes simplex virus type 1 (HSV-1) ICP4 genes specifying nonsense peptides.

Synthetic oligonucleotide linkers containing translational termination codons in all possible reading frames were inserted at various positions in the cloned gene encoding the herpes simplex virus type 1 (HSV-1) immediate-early regulatory protein, ICP4. It was determined that the amino-terminal 60 percent of the ICP4 gene was sufficient for trans-induction of a thymidine kinase promoter-CAT chimera (pTKCAT) and negative regulation of an ICP4 promoter-CAT chimera (pIE3CAT); however, it was relatively inefficient in complementing an ICP4 deletion mutant. The amino-terminal ninety amino acids do not appear to be required for infectivity as reflected by the replication competence of a mutant virus containing a linker insertion at amino acid 12. The size of the ICP4 molecule expressed from the mutant virus was consistent with translational restart at the next methionine codon corresponding to amino acid 90 of the deduced ICP4 amino acid sequence.     

Specificity of cotranslational amino-terminal processing of proteins in yeast

Polypeptides synthesized in the cytoplasm of eukaryotes are generally initiated with methionine, but N-terminal methionine is absent from most mature proteins. Many proteins are also N alpha-acetylated. The removal of N-terminal methionine and N alpha-acetylation are catalyzed by two enzymes during translation. The substrate preferences of the methionine aminopeptidase (EC 3.4.11.x) and N alpha-acetyltransferase (EC 2.3.1.x) have been partially inferred from the distribution of amino-terminal residues and/or mutations found for appropriate mature proteins, but with some contradictions. In this study, a synthetic gene corresponding to the mature amino acid sequence of the plant protein thaumatin, expressed in yeast as a nonexported protein, i.e., lacking a signal peptide, has been used to delineate the specificities of these enzymes with respect to the penultimate amino acid. Site-directed mutagenesis, employing synthetic oligonucleotides, was utilized to construct genes encoding each of the 20 amino acids following the initiation methionine codon, and each protein derivative was isolated and characterized with respect to its amino-terminal structure. All four possible N-terminal variants--those with and without methionine and those with and without N alpha-acetylation--were obtained. These results define the specificity of these enzymes in situ and suggest that the nature of the penultimate amino-terminal residue is the major determinant of their selectivity.     

Primary structure of human preangiotensinogen deduced from the cloned cDNA sequence

Cloned cDNA sequences for human preangiotensinogen have been isolated from a human liver cDNA library by hybridization with a restriction fragment derived from a previously cloned cDNA for rat preangiotensinogen. Analyses by nucleotide sequence determination, S1 nuclease mapping, and RNA blot hybridization indicate that human preangiotensinogen is encoded by two mRNAs that differ only in the length of the 3'-untranslated region. The deduced amino acid sequence shows that the mature angiotensinogen consists of 452 amino acid residues with the angiotensin sequence at its amino-terminal portion. Two potential initiation sites have been discussed. These are the methionine codon located at the position exactly corresponding to the initiation site of rat preangiotensinogen mRNA and an additional methionine codon positioned nearest the 5' end of the mRNA. The amino acid sequences starting at either of the initiation sites and preceding the angiotensin sequence constitute a large number of hydrophobic amino acid residues, thus representing the signal peptide characteristic of the secretory proteins. Human and rat preangiotensinogens show that 63.6% of the amino acid positions of the two proteins are identical. However, the amino-terminal portions directly distal to angiotensin I diverge markedly between the two proteins and differ in their possible glycosylation sites. These structural differences may contribute to the known species specificity exhibited by renin.     

The multiplicity of human pancreatic secretory trypsin inhibitor

Four forms of pancreatic secretory trypsin inhibitor (PSTI; A1, A2, B, and C) were purified from human pancreatic juice. According to sequence results, the primary structure of B was different from that reported earlier (Greene, L.J., et al. (1976) Method Enzymol. 45, 813-825) at two positions, i.e. Asn21----Asp21, Asp29----Asn29. A1 and A2 were deamidated forms of B judging from peptide mappings with Staphylococcus aureus V8 protease. Gln45 in B was replaced by Glu in A1 and Gln51 in B was replaced by Glu in A2. C was an inhibitor lacking five amino acid residues from the amino terminal of B. B and C inhibited human cationic trypsin activity stoichiometrically with similar dissociation constants, but A1 and A2 showed poorer trypsin inhibitory activity than B and C.     

Partition of trypsin and Kazal inhibitor in reaction mixtures with human serum.

The partition of trypsin and pancreatic secretory trypsin inhibitor (PSTI) in reaction mixtures with human serum was studied by electroimmunoassay and also by gel filtration on Sephadex G-200. The same pattern of trypsin complexes with alpha2-macroglobulin and alpha1-antitrypsin was observed in the presence or absence of PSTI. When sufficient trypsin was added to saturate the alpha2-macroglobulin, more complex with alpha1-antitrypsin was formed. A small amount of PSTI-trypsin complex was formed only when large amounts of trypsin and PSTI were present. The majority of PSTI was found in the fractions containing alpha2-macroglobulin, indicating the formation of a PSTI-trypsin-alpha2-macroglobulin complex. The remaining PSTI was eluted as free inhibitor. Increasing the added PSTI increased the fraction eluted as free inhibitor. alpha1-Antitrypsin and alpha2-macroglobulin appear to be much stronger than PSTI in their competition for trypsin in reaction mixtures of human serum, trypsin and PSTI.     

Human pancreatic secretory trypsin inhibitor (PSTI) produced in active form and secreted from Escherichia coli

As a basis for a protein design project, we decided to produce the human pancreatic secretory trypsin inhibitor (PSTI) in its active form. Total gene synthesis was carried out efficiently by (i) computer design of the gene fragments, (ii) synthesis of the oligodeoxynucleotides by the segmental support method, and (iii) assembly of double strands under optimized ligation conditions. Fusion to the ompA gene signal peptide led to secretion of processed PSTI in various constructions, with or without additional amino acids (aa) at the N-terminus. The secreted proteins (56 to 63 aa) were biologically active, suggesting that the three cysteine bridges were correctly formed. Surprisingly, after induction the product was found almost exclusively in the culture medium. Variants of PSTI with Asp or Asn at aa positions 21 and 29 [sequences published by Greene et al., Methods Enzymol. (1976) 813-825, and by Yamamoto et al., Biochem. Biophys. Res. Commun. (1985) 605-612] showed the same Ki for both human and porcine trypsin.     

Expression of recombinant proteins lacking methionine as N-terminal amino acid in plastids: human serum albumin as a case study

Removal of the N-terminal methionine of a protein could be critical for its function and stability. Post-translational modifications of recombinant proteins expressed in heterologous systems may change amino-terminal regions. We studied the expression of mature proteins lacking methionine as the N-terminal amino acid in tobacco chloroplasts, using human serum albumin (HSA) as an example. Two approaches were explored. First, we fused the Rubisco small subunit transit peptide to HSA. This chimeric protein was correctly processed in the stroma of the chloroplast and rendered the mature HSA. The second approach took advantage of the endogenous N-terminal methionine cleavage by methionine aminopeptidase. Study of this protein processing reveals a systematic cleavage rule depending on the size of the second amino acid. Analysis of several foreign proteins expressed in tobacco chloroplasts showed a cleavage pattern in accordance to that of endogenous proteins. This knowledge should be taken into account when recombinant proteins with N-terminus relevant for its function are expressed in plastids.     

On the cDNA's for two types of rat pancreatic secretory trypsin inhibitor

Two types of cDNA, which code for the two types of rat pancreatic secretory trypsin inhibitors (PSTIs), were cloned and sequenced. Both predicted amino acid sequences consisting of 79 amino acids, with the secretion signal peptide consisting of 18 and 23 amino acids for PSTI-I and PSTI-II, respectively. The nucleotide sequences were 91% homologous between the two cDNAs, but 68% and 65% homologous, respectively, when compared with human PSTI cDNA. Northern blot analyses showed that PSTI-I is expressed in the pancreas, whereas PSTI-II is expressed in the pancreas and the liver using the same promoter. Southern blot analyses suggested that both PSTI-I and PSTI-II genes are single copy genes per haploid genome. Duplication of rat PSTI gene seems to have occurred recently, after the divergence of humans and rats.     

Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation.

We isolated a cDNA encoding a functional human thrombin receptor by direct expression cloning in Xenopus oocytes. mRNA encoding this receptor was detected in human platelets and vascular endothelial cells. The deduced amino acid sequence revealed a new member of the seven transmembrane domain receptor family with a large amino-terminal extracellular extension containing a remarkable feature. A putative thrombin cleavage site (LDPR/S) resembling the activation cleavage site in the zymogen protein C (LDPR/I) was noted 41 amino acids carboxyl to the receptor's start methionine. A peptide mimicking the new amino terminus created by cleavage at R41 was a potent agonist for both thrombin receptor activation and platelet activation. "Uncleavable" mutant thrombin receptors failed to respond to thrombin but were responsive to the new amino-terminal peptide. These data reveal a novel signaling mechanism in which thrombin cleaves its receptor's amino-terminal extension to create a new receptor amino terminus that functions as a tethered ligand and activates the receptor.     

Reinvestigation of the disulfide bridge arrangement in human pro-opiomelanocortin N-terminal segment (hNT 1-76)

The cystine bridge structure of the amino-terminal fragment of human pro-opiomelanocortin has been reinvestigated. Highly purified amino-terminal fragment 1-76 was rapidly isolated from human pituitaries using only reverse-phase liquid chromatography (RP-HPLC). This peptide was then subjected to trypsin and V8-protease digestion and the products separated by RP-HPLC and subjected to amino acid and microsequence analysis. The results show that disulfide bridges link Cys-2 to Cys-24 and Cys-8 to Cys-20. Amino acid analysis and amino sugar determination confirm (i) the previously proposed sequence and (ii) the suggestion of the presence of two glycosylation sites in this molecule. These are most probably located at Thr-45 (O-glycosylation) and at Asn-65 (N-glycosylation).