Human Pancreatic Ribonuclease Research Articles

Cloning and cytotoxicity of a human pancreatic RNase immunofusion

Background: Immunotoxins based on plant and bacterial proteins are usually very immunogenic. Human ribonucleases could provide an alternative basis for the construction of less immunogenic reagents. Two members of the human RNase family, angiogenin and eosinophil-derived neurotoxin (EDN), have been fused to a single chain antibody against the transferrin receptor, which is known to be internalised by endocytosis. The fusion proteins proved to be very efficient inhibitors of protein synthesis using various cell lines. It is not yet known whether the side effects of angiogenin and EDN will compromise their potential use as immunotoxins. Objectives: The goal of this work was to construct a human immunotoxin with no harmful side effects. Bovine pancreatic ribonuclease has been shown to be as potent as ricin at abolishing protein synthesis on injection into oocytes. We therefore decided to clone its human analogue, which is fairly ubiquitous and per se non-toxic. An immunofusion of human pancreatic RNase with a single chain antibody against the transferrin receptor was tested for its ability to inhibit protein synthesis in three different human tumor cell lines. Study design: DNA coding for the human pancreatic RNase was cloned partially from a human fetal brain cDNA library and then completed by PCR using a human placental cDNA library as a template. The RNase gene was then fused with a DNA coding for an single chain antibody against the transferrin receptor (CD71). After expressing the fusion protein in E. coli, the gene product was isolated from inclusion bodies and tested for cytotoxicity. Results: This fusion protein inhibited the protein synthesis of three human tumor cell lines derived from a melanoma, a renal carcinoma and a breast carcinoma, with IC50s of 8, 5 and 10 nM, respectively. These values were comparable with those using a similar fusion protein constructed with eosinophil derived neurotoxin (EDN) as the toxic moiety (IC50s of 8, 1.2 and 3 nM, respectively). The slightly lower activities of the human pancreatic RNase-scFv (pancRNase-scFv) with two of the cell lines suggests that fewer molecules are reaching the cytoplasmic compartment, since it was twice as active as EDN-scFv in inhibiting the protein synthesis of a rabbit reticulocyte lysate. Conclusion: These results demonstrate that the human pancreatic RNase, which is expected to have a very low immunogenic potential in humans with no inherent toxicity, may be a potent cytotoxin for tumor cells after antibody targeting.

Human pancreatic ribonuclease--deletion of the carboxyl-terminal EDST extension enhances ribonuclease activity and thermostability.

Mammalian ribonucleases constitute one of the fastest evolving protein families in nature. The addition of a four-residue carboxyl-terminal tail: Glu-Asp-Ser-Thr (EDST) in human pancreatic ribonuclease (HPR) in comparison with bovine pancreatic RNase (RNase A) could have adaptive significance in humans. We have cloned and expressed human pancreatic ribonuclease in Escherichia coli to probe the influence of the four-residue extension and neighboring C-terminal residues on the biochemical properties of the enzyme. Removal of the C-terminal extension from HPR yielded an enzyme, HPR-(1-124)-peptide, with enhanced ability to cleave poly(C). HPR-(1-124)-peptide also exhibited a steep increase in thermal stability mimicking that known for RNase A. Wild-type HPR had significantly low thermal stability compared to RNase A. The study identifies the C-terminal boundary in the human pancreatic ribonuclease required for efficient catalysis.

Inhibition of HIV-1 Production and Selective Degradation of Viral RNA by an Amphibian Ribonuclease

Ribonucleases appear to have physiologic roles in host defense against cancer, viruses, and other parasites. Previously it was shown that select ribonucleases added to cells concurrently with virions blocked human immunodeficiency virus, type I (HIV-1) infection of H9 cells. We now report that a ribonuclease homologous to RNase A, named onconase, inhibits virus replication in chronically HIV-1-infected human cells without killing the virally infected cell. Examining the mechanism of this inhibition shows that onconase enters the infected cells and degrades HIV-1 RNA without degrading ribosomal RNA or the three different cellular messenger RNAs analyzed. The homologous human pancreatic RNase lacks anti-viral activity. Comparing recombinant forms of onconase and a onconase-human RNase chimera shows that the N-terminal 9 amino acids and the pyroglutamyl residue of onconase are required for full anti-viral activity. Thus extracellular ribonucleases can enter cells, metabolize select RNAs, and inhibit HIV virion production within viable replicating cells.

Production of Human Pancreatic Ribonuclease inSaccharomyces cerevisiaeandEscherichia coli

Human pancreatic ribonuclease (HP-RNase) has considerable promise as a therapeutic agent. Structure–function analyses of HP-RNase have been impeded by the difficulty of obtaining the enzyme from its host. Here, a gene encoding HP-RNase was designed, synthesized, and inserted into two expression vectors that then direct the production of HP-RNase inSaccharomyces cerevisiae(fused to either an unmodified or a modified α-factor pre–pro segment) orEscherichia coli(fused to the pelB signal sequence). HP-RNase produced inS. cerevisiaewas secreted into the medium as an active enzyme, isolable at 0.1–0.2 mg/liter of culture. This isolate was heterogeneous due to extensive glycosylation and incomplete maturation of the pre–pro segment. HP-RNase produced inE. coliwith the pET expression system was purified from the insoluble fraction of the cell lysate. Renaturation of the reduced and denatured protein produced active, homogeneous enzyme recoverable at 1 mg/liter of culture. The N terminus of the HP-RNase produced from the bacterial expression system was processed fullyin vivo.The yeast system, combined with techniques that allow detection of picograms of ribonuclease activity, offers a sensitive probe for studies of post-translational modification and secretory targeting in eukaryotic cells. The bacterial system enables studies both to reveal new structure–function relationships in ribonucleases and to evaluate the use of HP-RNase as a cytotoxin that is tolerated by the human immune system.

Role of the N Terminus in RNase A Homologues: Differences in Catalytic Activity, Ribonuclease Inhibitor Interaction and Cytotoxicity

A number of biochemical properties differ dramatically among homologues within the pancreatic ribonuclease superfamily. Human pancreatic ribonuclease (hRNase) has high enzyme activity, extreme sensitivity to ribonuclease inhibitor (RI) and is non-toxic, whereas a homologous RNase from frog eggs, called onconase, has much lower enzyme activity, is not sensitive to RI and is cytotoxic to cancer cell lines and animals. To explore the structural basis of these differences among members in the RNase family we synthesized genes for onconase, hRNase, a mutant onconase (K9Q) and onconase-hRNase N-terminal hybrids and expressed the proteins in Escherichia coliwith final yields of 10 to 50 mg per liter of culture after purification. A recombinant version of onconase with an N-terminal methionine instead of the native pyroglutamyl residue had decreased cytotoxicity and enzyme activity. Cleavage of the recombinant onconase Met − 1 residue, and cyclization of the Gln1 residue to reform the pyroglutamyl N terminus, reconstituted cytotoxicity and enzyme activity. Thus a unique role of the pyroglutamyl residue in the active site of amphibian RNases is indicated. Replacement of one to nine residues of onconase with the homologous residues of hRNase increased the enzymatic activity against most of the substrates tested with a simultaneous shift in the enzyme specificity from high preference for poly(U) to slight preference for poly(C). Cytotoxicity of the chimera decreased, dissociating cytotoxicity from enzymatic activity. The molecular basis for the low binding affinity of onconase for RI has been examined experimentally with the recombinant RNases and by fitting onconase and RNase A structures to the coordinates from the recently published RNase A-RI complex.

Recombinant Human Pancreatic Ribonuclease Produced in E. coli : Importance of the Amino-Terminal Sequence

Human pancreatic ribonuclease I (hRNase 1) in the mature form has been produced in E.coli using T7 expression system. The recombinant hRNase 1 protein was solubilized from the inclusion bodies, refolded in glutathione redox system, and purified through chromatographic procedures by utilizing cation-exchange and reversed-phase columns. The ribonucleolytic activity of recombinant hRNase 1 was examined on yeast RNA and cytidylyl-3′,5′-adenosine revealing the distinctive ribonucleolytic activity. The activity was perfectly inhibited by human placental RNase inhibitor. Truncation of 7 amino acid residues in the amino-terminal sequence resulted in much reduction in ribonucleolytic activity and in affinity to human placental RNase inhibitor with the disintegration of secondary structures of the protein observed by circular dichroism spectra. The present study has revealed the important contribution of the amino-terminal sequence of hRNase I to the characteristics of the protein.

Cloning and Expression of An Engineered Human Pancreatic Ribonuclease

The coding sequence for the human pancreatic ribonuclease (HP-RNase) gene has been obtained from genomic DNA extracted from buccal epithelial cells. In order to direct the expression of the recombinant human pancreatic enzyme to the periplasmic space of E.coli, the bovine pancreatic RNase signal sequence has been fused 5' to the human gene. Initial attempts to express the recombinant enzyme were not successful, consequently site-directed mutagenesis tehniques were used to genetically engineer the HP-RNase gene to enable expression in E.coli. The resultant engineered enzyme shows similar kinetic characteristics to the homologous bovine enzyme.

Nucleotide sequence encoding human pancreatic ribonuclease

A cDNA coding for human pancreatic ribonuclease was isolated from a pancreas cDNA library and sequenced. This cDNA (1620 bp) includes an entire open reading frame encoding mature protein (128 aa) following a signal peptide (28 aa) as well as 5′-and 3′-untranslated regions.

Expression in mammalian cells, purification and characterization of recombinant human pancreatic ribonuclease.

A synthetic cDNA coding for human pancreatic RNase, equipped with a secretion signal sequence, was cloned and stably expressed in Chinese hamster ovary cells. The recombinant RNase, secreted into the culture medium, was purified and characterized. It was found to be indistinguishable, by structural and catalytic parameters, from the enzyme isolated from human pancreas. Furthermore, the glycosylated forms were separated from the non-glycosylated form. Up until now, human RNases have been isolated only in small amounts from autopic specimens. This has hindered the exploitation of a human RNase for the construction of immunotolerated immunotoxins. On the other hand, the availability of an effective system for the expression of a human RNase may render feasible the transfer, by protein engineering, of the interesting pharmacological actions of non-human RNase [1993 Trends Cell Biol. 3, 106-109] to an immunotolerated, human RNase.

Eosinophil-derived neurotoxin and human liver ribonuclease. Identity of structure and linkage of neurotoxicity to nuclease activity.

Eosinophil-derived neurotoxin (EDN) and human liver RNase were found to be indistinguishable from each other but distinct from the pancreatic ribonucleases in their nucleolytic activity on polynucleotides or small defined substrates. Antibodies to EDN and liver RNase showed identical cross-reactivities in assays of nuclease inhibition and in a radioimmunoassay. In each instance, EDN and liver RNase were easily distinguished from bovine or human pancreatic RNase. When injected intrathecally into rabbits, 5-10 micrograms of EDN or liver RNase each was neurotoxic as judged by induction of the Gordon phenomenon. Human pancreatic RNase was less neurotoxic, and up to 20-fold higher levels of bovine pancreatic RNase showed no effect. Treatment of EDN, liver RNase, and eosinophil cationic protein with iodoacetic acid at pH 5.5 resulted in inactivation of their RNase activity and also destroyed their neurotoxicity. EDN conformation was not greatly affected by iodoacetate treatment since interaction of the modified protein with antibodies was only slightly altered. We conclude that RNase activity is necessary but not sufficient to induce neurotoxic action.

The primary structure of langur (Presbytis entellus) pancreatic ribonuclease: adaptive features in digestive enzymes in mammals.

The primary structure of pancreatic ribonuclease from langur (Presbytis entellus) has been determined. This sequence differs from that of human pancreatic ribonuclease at 14 (11%) of the amino acid positions. Eight of these 14 differences involve changes of charge, with the langur enzyme having five fewer positive charges than the human enzyme. The difference in charge between human and langur ribonuclease may be an adaptation to the different requirements for a nondigestive and a digestive role, respectively. A number of similarities in expression, gene duplications, and properties between mammalian ribonucleases and lysozymes have been observed, indicating similar adaptations in both enzyme systems.

Eosinophil cationic protein cDNA. Comparison with other toxic cationic proteins and ribonucleases.

Human eosinophil granules contain several basic proteins including eosinophil cationic protein (ECP), eosinophil-derived neurotoxin (EDN) and major basic protein (MBP). ECP and MBP are potent helminthotoxins while EDN is less so. Both ECP and EDN possess neurotoxic and ribonuclease activities. A clone representing ECP mRNA was isolated from an eosinophil lambda ZAP cDNA library. The cDNA sequence codes for a preprotein of 160 amino acids and a protein of 133 amino acids, the amino terminus of which is identical to the known partial amino acid sequence of ECP. The ECP nucleotide sequence shows similarity to EDN, rat pancreatic ribonuclease, and human angiogenin; all are members of the ribonuclease gene superfamily. Although the deduced amino acid sequence of ECP shares identical active site and substrate binding site residues with EDN, angiogenin, and human pancreatic ribonuclease, the ribonuclease activity of ECP is 50 to 100 times less than that of EDN possibly because of the lack of a positively charged residue at human pancreatic ribonuclease position 122. The calculated isoelectric point (10.8), electronic charge (14.5), and cationic charge distribution of ECP are different from those of EDN but similar to those of MBP, which may account in part for the greater helminthotoxic activity of ECP when compared to EDN. These data suggest that ECP and EDN are derived from a common ancestral ribonuclease gene and that ECP has evolved into a potent helminthotoxin similar in some respects to MBP, while losing much of its ribonuclease activity.

Human eosinophil cationic protein. Molecular cloning of a cytotoxin and helminthotoxin with ribonuclease activity.

We have isolated a 725-bp full-length cDNA clone for the human eosinophil cationic protein (ECP). ECP is a small, basic protein found in the matrix of the eosinophil's large specific granule that has cytotoxic, helminthotoxic, and ribonuclease activity, and is a member of the ribonuclease multigene family. The cDNA sequence shows 89% sequence identity with that reported for the related granule protein, eosinophil-derived neurotoxin (EDN). The open reading frame encodes a previously unidentified 27-amino acid leader sequence preceding a 133-residue mature ECP polypeptide with a molecular mass of 15.6 kD. The encoded amino acid sequence of ECP shows 66% identity to that of EDN and 31% identity to that of human pancreatic ribonuclease, including conservation of the essential structural cysteine and cataytic lysine and histidine residues. mRNA for ECP was detected in eosinophil-enriched peripheral granulocytes and in a subclone of the promyelocytic leukemia line, HL-60, induced toward eosinophilic differentiation with IL-5. No ECP mRNA was detected in uninduced HL-60 cells, or in HL-60 cells induced toward monocytic differentiation with vitamin D3 or toward neutrophilic differentiation with DMSO. In contrast, mRNA for EDN was detected in uninduced HL-60 cells and was upregulated in HL-60 cells induced with DMSO. Despite similarities in sequence and cellular localization, these results suggest that ECP and EDN are subject to different regulatory mechanisms.

Purification and characterization of a ribonuclease from human liver.

The major ribonuclease of human liver has been isolated in a four-step procedure. The protein appears homogeneous by several criteria. The amino acid composition and the amino-terminal sequence of the enzyme indicate that the protein is related to human pancreatic ribonuclease and to angiogenin, and that it may be identical with an eosinophil-derived neurotoxin and to a ribonuclease that has been isolated from urine. The catalytic activity of the liver ribonuclease and its sensitivity to iodoacetic acid inactivation also relate the enzyme to the pancreatic RNases, but the liver protein is clearly differentiated by immunological measurements. Antibodies to the liver ribonuclease inhibit its activity, but not that of the human pancreatic enzyme; cross-reactivity in a radioimmunological assay is small but measurable. Immunochemical measurements have been used to examine the distribution of the liver-type protein in other tissues. Inhibition of enzyme activity by anti-liver ribonuclease shows that a cross-reactive enzyme is predominant in extracts of spleen and is a significant component in kidney preparations, while the liver-type protein is almost absent in brain or pancreas homogenates. Cross-reactive ribonuclease is present in serum, but levels are not correlated with any of the disease states examined.

Enzymatically active angiogenin/ribonuclease A hybrids formed by peptide interchange.

The primary structures of the blood vessel inducing protein human angiogenin and human pancreatic ribonuclease (RNase) are 35% identical. Angiogenin catalyzes the limited cleavage of ribosomal RNA (18 and 28 S), yielding a characteristic pattern of polynucleotide products, but shows no significant activity toward conventional pancreatic RNase substrates [Shapiro, R., Riordan, J. F., & Vallee, B. L. (1986) Biochemistry 25, 3527-3532]. Angiogenin/RNase hybrid enzymes--wherein particular regions of primary structure in RNase are replaced by the corresponding segments of angiogenin--serve to explore the structural features underlying angiogenin's characteristic activities. Herein we show that synthetic angiogenin peptides, Ang(1-21) and Ang(108-123), form noncovalent complexes with inactive fragments of bovine RNase A--RNase(21-124) (i.e., S-protein) and RNase(1-118), respectively--with regeneration of activity toward conventional RNase substrates. Maximal activities for the Ang(1-21)/S-protein complex (Kd = 1.0 microM) are 52%, 45%, and 15% toward cytidine cyclic 2',3'-phosphate, cytidylyl(3'----5')adenosine, and yeast RNA, respectively. In contrast, activities of the RNase(1-118)/Ang(108-123) hybrid (Kd = 25 microM) are 1-2 orders of magnitude lower toward cyclic nucleotides and dinucleoside phosphates. However, substitution of phenylalanine for Leu-115 in Ang(108-123) increases activity up to 100-fold. Both His-13 and His-114 in the angiogenin peptides are required for activity since their substitution by alanine yields inactive complexes. Importantly, the pattern of polynucleotide products formed during cleavage of ribosomal RNA by the Ang(1-21)/S-protein hybrid shows a striking resemblance to that formed by angiogenin, demonstrating that the hybrid retains features of both angiogenin and RNase A.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunohistochemical Localization of Pancreatic Ribonuclease Antigen in Human Tissues by the Peroxidase-Antiperoxidase Technique

Human pancreatic ribonuclease (RNase) antigen was localized in tissue sections by the unlabeled antibody-peroxidase antiperoxidase (PAP) method, using polyclonal rabbit antibody to human pancreatic RNase. The antigen was demonstrated in the cytoplasm of pancreatic acinar cells, tissue macrophages and Kupffer cells. Pancreatic RNase antigen was not detected in hepatocytes nor in splenic cells other than macrophages, indicating antigenic nonidentity between pancreatic and liver/spleen ribonucleases. The kidney showed differential staining, with pancreatic RNase antigen present in proximal tubules and absent from distal tubules, collecting ducts, and glomeruli. No antigen was detected in skeletal, cardiac or smooth muscle, epidermis, or germ cells. Preliminary results showed that pancreatic RNase antigen was present in small quantity in some pancreatic adenocarcinomas. This method should be useful in combination with other techniques for determining the distribution of RNase antigen in normal and diseased tissues.

Cloning and sequencing of the intronless gene for angiogenin, a human angiogenesis factor

Human cDNAs coding for angiogenin, a human tumor derived angiogenesis factor, were isolated from a cDNA library prepared from human liver poly(A) mRNA employing a synthetic oligonucleotide as a hybridization probe. The largest cDNA insert (697 base pairs) contained a short 5'-noncoding sequence followed by a sequence coding for a signal peptide of 24 (or 22) amino acids, 369 nucleotides coding for the mature protein of 123 amino acids, a stop codon, a 3'-noncoding sequence of 175 nucleotides, and a poly(A) tail. The gene coding for human angiogenin was then isolated from a genomic lambda Charon 4A bacteriophage library employing the cDNA as a probe. The nucleotide sequence of the gene and the adjacent 5'- and 3'-flanking regions (4688 base pairs) was then determined. The coding and 3'-noncoding regions of the gene for human angiogenin were found to be free of introns, and the DNA sequence for the gene agreed well with that of the cDNA. The gene contained a potential TATA box in the 5' end in addition to two Alu repetitive sequences immediately flanking the 5' and 3' ends of the gene. The third Alu sequence was also found about 500 nucleotides downstream from the Alu sequence at the 3' end of the gene. The amino acid sequence of human angiogenin as predicted from the gene sequence was in complete agreement with that determined by amino acid sequence analysis. It is about 35% homologous with human pancreatic ribonuclease, and the amino acid residues that are essential for the activity of ribonuclease are also conserved in angiogenin. This provocative finding is thought to have important physiological implications.

The amino acid sequence of human pancreatic ribonuclease

The primary structure of human ( Homo sapiens) pancreatic ribonuclease has been determined by automatic sequencing of the native protein and by analysis of peptides obtained by cleavage with proteolytic enzymes, cyanogen bromide, and hydroxylamine. The following sequence was deduced: ▪ Human pancreatic ribonuclease differs at 37 positions from bovine pancreatic ribonuclease. In addition the human enzyme has three more residues at the C-terminus. About half of the enzyme molecules contain carbohydrate attached to the sequence Asn-Met-Thr (34–36). Two other Asn-X-Ser Thr sequences are carbohydrate free. Human pancreatic ribonuclease contains many positively charged residues, especially near the N-terminus, while negatively charged residues are more concentrated near the C-terminus.

Radioimmunoassays for two types of human urinary ribonucleases: differential determination of ribonucleases in human serum

We developed radioimmunoassays for two types of human urinary ribonucleases. The assays are sensitive, reproducible and specific. One human urinary ribonuclease (RNase U l) showed strong immunological identity with human pancreatic ribonuclease, and the other ribonuclease (RNase Us) showed strong immuno crossreactivity with human liver ribonuclease. There was no immuno cross-reactivity between these two urinary ribonucleases. Serum immunoreactive RNase U l was eluted as four peaks on phosphocellulose chromatography, whereas immunoreactive RNase Us was eluted as a single peak. Serum content of immunoreactive RNase U l was 354 ± 105 ng/ml (mean ± SD) in normal individuals, and that of immunoreactive RNase Us was 15.9 ± 5.7 ng/ml (mean ± SD). No correlation was demonstrated between these two ribonuclease contents in serum.

Purification, characterization and development of radioimmunoassay of human liver ribonuclease

Human liver ribonuclease (RNase) was purified 3600-fold into an electrophoretically homogeneous state by column chromatography on phosphocellulose, gel filtration, poly(G) affinity chromatography, and heparin affinity chromatography. The molecular weight of the RNase estimated by SDS disc electrophoresis was 19500. RNase was a heat- and pH-stable protein, and optimum activity was obtained at pH 7.0. The radioimmunoassay (RIA) for human liver RNase has been developed and the assay was shown to be sensitive (20 ng/ml), reproducible and specific. A good parallel relationship was observed between the standard curve and the dilution curves for serum and urine. No cross-reactivity was demonstrated between human liver and pancreatic RNase (less than 1%). In 44 normal subjects, the mean serum concentration of liver RNase determined by the RIA was found to be 99.4 ng/ml (SD ± 66.3).