Human Pancreatic Ribonuclease Research Articles

The Solution Structure and Dynamics of Human Pancreatic Ribonuclease Determined by NMR Spectroscopy Provide Insight into Its Remarkable Biological Activities and Inhibition

Human pancreatic ribonuclease (RNase 1) is expressed in many tissues; has several important enzymatic and biological activities, including efficient cleavage of single-stranded RNA, double-stranded RNA and double-stranded RNA–DNA hybrids, digestion of dietary RNA, regulation of vascular homeostasis, inactivation of the HIV, activation of immature dendritic cells and induction of cytokine production; and furthermore shows potential as an anti-tumor agent. The solution structure and dynamics of uncomplexed, wild-type RNase 1 have been determined by NMR spectroscopy methods to better understand these activities. The family of 20 structures determined on the basis of 6115 unambiguous nuclear Overhauser enhancements is well resolved (pairwise backbone RMSD = 1.07 Å) and has the classic RNase A type of tertiary structure. Important structural differences compared with previously determined crystal structures of RNase 1 variants or inhibitor-bound complexes are observed in the conformation of loop regions and side chains implicated in the enzymatic as well as biological activities and binding to the cytoplasmic RNase inhibitor. Multiple side chain conformations observed for key surface residues are proposed to be crucial for membrane binding as well as translocation and efficient RNA hydrolysis. 15N– 1H relaxation measurements interpreted with the standard and our extended Lipari–Szabo formalism reveal rigid regions and identify more dynamic loop regions. Some of the most dynamic areas are key for binding to the cytoplasmic RNase inhibitor. This finding and the important differences observed between the structure in solution and that bound to the inhibitor are indications that RNase 1 to inhibitor binding can be better described by the “induced fit” model rather than the rigid “lock-into-key” mechanism. Translational diffusion measurements reveal that RNase 1 is predominantly dimeric above 1 mM concentration; the possible implications of this dimeric state for the remarkable biological properties of RNase 1 are discussed.

Development of Genotyping Methods for Single Nucleotide Polymorphism in the Human Pancreatic Ribonuclease Gene (RNASE1) and Their Application to Population Studies

Two potential single nucleotide polymorphisms [SNPs; rs1804215 (G979T) and rs11545379 (G1169T)] have been identified in the human pancreatic ribonuclease, RNase 1, gene (RNASE1) that could give rise to an amino acid substitution in the protein, but relevant population data are not available. We have developed genotyping methods for each SNP using the mismatched PCR-restriction fragment length polymorphism technique. These methods are advantageous in comparison with other SNP genotyping methods because they are technically simpler and do not require specialized instruments. We applied these genotyping methods to examine the genotype distribution of each SNP in four populations, including Japanese populations living in two prefectures, an Ovambo population, and a Turkish population. In all the populations studied, however, only a single genotype for each SNP was found. Therefore, irrespective of differences in ethnic groups, RNASE1 might show markedly low heterogeneity in its genetic structure with regard to these SNPs.

Altered Glycosylation in Tumours Focused to Cancer Diagnosis

The lack of specific and sensitive tumour markers for early detection of cancer is driving a search for new approaches that could identify biomarkers. Markers are needed to alert clinicians at the early stages of tumourogenesis, before the cancer has metastasized, when the therapeutic drugs are more effective. Most tumour markers currently used in clinics are serum glycoproteins, frequently highly glycosylated mucins. Typically, the disease marker is the protein and not the glycan moiety of the corresponding glycoprotein or mucin. The increasing knowledge of the role of glycans in cancer suggests that further studies may assist both in determining their role in every step of tumour progression, and in the design of new therapeutic and diagnosic approaches. Detection of the altered glycans in serum tumour glycoproteins could be a way to achieve specificity in tumour detection. In this review, we focus on the glycan changes of two serum glycoproteins, prostate specific antigen - currently used as a tumour marker of prostate cancer - and human pancreatic ribonuclease in pancreatic adenocarcinoma. The detection of glycan changes, associated with subsets of glycoforms in serum glycoproteins that are specific to the tumour situation, could be the basis for developing more specific biomarkers.

Human pancreatic ribonuclease presents higher endonucleolytic activity than ribonuclease A

Analyzing the pattern of oligonucleotide formation induced by HP-RNase cleavage shows that the enzyme does not act randomly and follows a more endonucleolytic pattern when compared to RNase A. The enzyme prefers the binding and cleavage of longer substrate molecules, especially when the phosphodiester bond that is broken is 8–11 nucleotides away from at least one of the ends of the substrate molecule. This more endonucleolytic pattern is more appropriate for an enzyme with a regulatory role. Deleting two positive charges on the N-terminus (Arg4 and Lys6) modifies this pattern of external/internal phosphodiester bond cleavage preference, and produces a more exonucleolytic enzyme. These residues may reinforce the strength of a non-catalytic secondary phosphate binding (p 2) or, alternatively, constitute a new non-catalytic phosphate binding subsite (p 3).

Cytotoxic Ribonucleases: The Dichotomy of Coulombic Forces

Cells tightly regulate their contents. Still, nonspecific Coulombic interactions between cationic molecules and anionic membrane components can lead to adventitious endocytosis. Here, we characterize this process in a natural system. To do so, we create variants of human pancreatic ribonuclease (RNase 1) that differ in net molecular charge. By conjugating a small-molecule latent fluorophore to these variants and using flow cytometry, we are able to determine the kinetic mechanism for RNase 1 internalization into live human cells. We find that internalization increases with solution concentration and is not saturable. Internalization also increases with time to a steady-state level, which varies linearly with molecular charge. In contrast, the rate constant for internalization (t1/2 = 2 h) is independent of charge. We conclude that internalization involves an extracellular equilibrium complex between the cationic proteins and abundant anionic cell-surface molecules, followed by rate-limiting internalization. The enhanced internalization of more cationic variants of RNase 1 is, however, countered by their increased affinity for the cytosolic ribonuclease inhibitor protein, which is anionic. Thus, Coulombic forces mediate extracellular and intracellular equilibria in a dichotomous manner that both endangers cells and defends them from the potentially lethal enzymatic activity of ribonucleases.

Role of unique basic residues of human pancreatic ribonuclease in its catalysis and structural stability

Human pancreatic ribonuclease (HPR) and bovine RNase A belong to the RNase A superfamily and possess similar key structural and catalytic residues. Compared to RNase A, HPR has six extra non-catalytic basic residues and high double-stranded RNA (dsRNA) cleavage activity. We mutated four of these basic residues, K6, R32, K62, and K74 to alanine and characterized the variants for function and stability. Only the variant K74A had an altered secondary structure. Whereas R32A and K62A had full catalytic activity, the mutants K6A and K74A had reduced activity on both ssRNA and dsRNA. The mutations of K62 and K74 resulted in reduction in protein stability and DNA double helix unwinding activity of HPR; while substitutions of K6 and R32 did not affect either the stability or helix unwinding activity. The reduced catalytic and DNA melting activities of K74A mutant appear to be an outcome of its altered secondary structure. The basic residues studied here, appear to contribute to the overall stability, folding, and general catalytic activity of HPR.

'Crystal lattice engineering,' an approach to engineer protein crystal contacts by creating intermolecular symmetry: crystallization and structure determination of a mutant human RNase 1 with a hydrophobic interface of leucines.

A protein crystal lattice consists of surface contact regions, where the interactions of specific groups play a key role in stabilizing the regular arrangement of the protein molecules. In an attempt to control protein incorporation in a crystal lattice, a leucine zipper-like hydrophobic interface (comprising four leucine residues) was introduced into a helical region (helix 2) of the human pancreatic ribonuclease 1 (RNase 1) that was predicted to form a suitable crystallization interface. Although crystallization of wild-type RNase 1 has not yet been reported, the RNase 1 mutant having four leucines (4L-RNase 1) was successfully crystallized under several different conditions. The crystal structures were subsequently determined by X-ray crystallography by molecular replacement using the structure of bovine RNase A. The overall structure of 4L-RNase 1 is quite similar to that of the bovine RNase A, and the introduced leucine residues formed the designed crystal interface. To characterize the role of the introduced leucine residues in crystallization of RNase 1 further, the number of leucines was reduced to three or two (3L- and 2L-RNase 1, respectively). Both mutants crystallized and a similar hydrophobic interface as in 4L-RNase 1 was observed. A related approach to engineer crystal contacts at helix 3 of RNase 1 (N4L-RNase 1) was also evaluated. N4L-RNase 1 also successfully crystallized and formed the expected hydrophobic packing interface. These results suggest that appropriate introduction of a leucine zipper-like hydrophobic interface can promote intermolecular symmetry for more efficient protein crystallization in crystal lattice engineering efforts.

Inhibition of Human Pancreatic Ribonuclease by the Human Ribonuclease Inhibitor Protein

The ribonuclease inhibitor protein (RI) binds to members of the bovine pancreatic ribonuclease (RNase A) superfamily with an affinity in the femtomolar range. Here, we report on structural and energetic aspects of the interaction between human RI (hRI) and human pancreatic ribonuclease (RNase 1). The structure of the crystalline hRI·RNase 1 complex was determined at a resolution of 1.95 Å, revealing the formation of 19 intermolecular hydrogen bonds involving 13 residues of RNase 1. In contrast, only nine such hydrogen bonds are apparent in the structure of the complex between porcine RI and RNase A. hRI, which is anionic, also appears to use its horseshoe-shaped structure to engender long-range Coulombic interactions with RNase 1, which is cationic. In accordance with the structural data, the hRI·RNase 1 complex was found to be extremely stable ( t 1/2 = 81 days; K d = 2.9 × 10 − 16 M). Site-directed mutagenesis experiments enabled the identification of two cationic residues in RNase 1, Arg39 and Arg91, that are especially important for both the formation and stability of the complex, and are thus termed “electrostatic targeting residues”. Disturbing the electrostatic attraction between hRI and RNase 1 yielded a variant of RNase 1 that maintained ribonucleolytic activity and conformational stability but had a 2.8 × 10 3-fold lower association rate for complex formation and 5.9 × 10 9-fold lower affinity for hRI. This variant of RNase 1, which exhibits the largest decrease in RI affinity of any engineered ribonuclease, is also toxic to human erythroleukemia cells. Together, these results provide new insight into an unusual and important protein–protein interaction, and could expedite the development of human ribonucleases as chemotherapeutic agents.

Glycosylation of serum ribonuclease 1 indicates a major endothelial origin and reveals an increase in core fucosylation in pancreatic cancer

Human pancreatic ribonuclease 1 (RNase 1) is a glycoprotein expressed mainly by the pancreas and also found in endothelial cells. The diagnosis of pancreatic cancer (PaC) remains difficult and therefore the search for sensitive and specific markers is required. Previous studies showed that RNase 1 from human healthy pancreas contained only neutral glycans, whereas RNase 1 from PaC cell lines contained sialylated structures. To determine whether these glycan tumor cell-associated changes were also characteristic of serum RNase 1 and could be used as a marker of PaC, we have analyzed the glycosylation of serum RNase 1. The origin of serum RNase 1 was also investigated. Serum RNase 1 from two PaC patients and two controls was purified and the glycans analyzed by high-performance liquid chromatography (HPLC)-based sequencing and mass spectrometry. Although normal and tumor serum RNase 1 contained the same glycan structures, there was an increase of 40% in core fucosylation in the main sialylated biantennary glycans in the PaC serum RNase 1. This change in proportion would be indicative of a subset of tumor-associated glycoforms of RNase 1, which may provide a biomarker for PaC. Two-dimensional electrophoresis of the RNase 1 from several endothelial cell lines, EA.hy926, human umbilical vein endothelial cells (HUVEC), human mammary microvessel endothelial cells (HuMMEC), and human lung microvessel endothelial cells (HuLEC), showed basically the same pattern and was also very similar to that of serum RNase 1. RNase 1 from EA.hy926 was then purified and presented a glycosylation profile very similar to that from serum RNase 1, suggesting that endothelial cells are the main source of this enzyme.

Intracellular route and mechanism of action of ERB-hRNase, a human anti-ErbB2 anticancer immunoagent

Human anti-ErbB2 immunoRNase with Erbicin fused to HP-RNase (ERB-hRNase) is a fully human immunoRNase made up of human pancreatic RNase fused to a human anti-ErbB2 scFv. It binds selectively with high affinity to ErbB2-positive cells, and specifically inhibits their proliferation, in vitro and in vivo. An investigation of its mechanism of action and its intracellular destination has revealed that ERB-hRNase depends on its RNase activity for cytotoxic action; it reaches the cytosol directly from the endosomal compartment; it is inhibited by the cytosolic RNase inhibitor (cRI), but the levels that ERB-hRNase reaches in the cytosol neutralize cRI, thus inducing cell death by apoptosis.

New anti-CD30 human pancreatic ribonuclease-based immunotoxin reveals strong and specific cytotoxicity in vivo

Although the therapy of Hodgkin lymphoma and anaplastic large cell lymphoma has been considerably improved during the last decades, high therapeutic toxicity, relapses, secondary tumors, and primary treatment failure(s) occur. Both malignancies are well suited for CD30-targeted immunotherapy because of their strong CD30 expression. We constructed an immunotoxin composed of a single chain variable fragment of a CD30 antibody fused to the human pancreatic ribonuclease, showing CD30-specific binding and ribonucleolytic activity resistant to the inhibitor RNasin. This immunotoxin revealed CD30-specific anti-tumor activity in BALB/c mice that were challenged with CD30-positive or CD30-negative syngeneic tumor cells.

Anti‐tumor effect in an in vivo model by human‐derived pancreatic RNase with basic fibroblast growth factor insertional fusion protein through antiangiogenic properties

It is thought that the export of angiogenic fibroblast growth factors (FGF) from tumors may be involved in the onset of tumor angiogenesis. To create a new active targeting drug that inhibits the tumor angiogenic process without toxicities to normal cells, human basic FGF (h-bFGF) was inserted genetically into the Gly89 position of cross-linked RNase1 (the ribonuclease inhibitor protein [RI] binding site of cross-linked human pancreatic RNase) to prevent stereospecific binding to RI. The resultant insertional-fusion protein (CL-RFN89) was active both as h-bFGF and as RNase1. Furthermore, it acquired an additional ability of evading RI through steric blockade of RI binding caused by the fused h-bFGF domain. In the present study, the effect of the resultant protein, CL-RFN89, on the antitumor response though its antiangiogenic properties was investigated in an in vivo model. Continuous systemic treatment with CL-RFN89 significantly inhibited the growth of human A431 squamous cell carcinomas in vivo. Seven days of treatment with CL-RFN89 resulted in a 58.2% inhibition of tumor growth compared with control mice (P < 0.0001). Furthermore, immunohistochemistry using a rat antimouse CD31 antibody showed that treatment with CL-RFN89 reduced tumor vascularization. These findings identify CL-RFN89 as a potent systemic inhibitor of tumor growth as a result of its antiangiogenic properties. This protein appears to be a new systemic antitumor agent.

Characterization of the dimerization process of a domain‐swapped dimeric variant of human pancreatic ribonuclease

It has been previously reported that the structure of a human pancreatic ribonuclease variant, namely PM8, constitutes a dimer by the exchange of an N-terminal domain, although in an aqueous solution it is found mainly as a monomer. First, we investigated the solution conditions that favour the dimerization of this variant. At 29 degrees C in a 20% (v/v) ethanol buffer, a significant fraction of the protein is found in dimeric form without the appearance of higher oligomers. This dimer was isolated by size-exclusion chromatography and the dimerization process was studied. The dissociation constant of this dimeric form is 5 mm at 29 degrees C. Analysis of the dependence of the dimerization process on the temperature shows that unlike bovine pancreatic ribonuclease, a decrease in the temperature shifts the monomer-dimer equilibrium to the latter form. We also show that a previous dissociation of the exchangeable domain from the main protein body does not take place before the dimerization process. Our results suggest a model for the dimerization of PM8 that is different to that postulated for the dimerization of the homologous bovine pancreatic ribonuclease. In this model, an open interface is formed first and then intersubunit interactions stabilize the hinge loop in a conformation that completely displaces the equilibrium between nonswapped and swapped dimers to the latter one.

Cytotoxicity of human RNase-based immunotoxins requires cytosolic access and resistance to ribonuclease inhibition

Immunotoxins are targeted therapeutics designed to kill cancer cells. The targeting moiety of an immunotoxin selectively binds to a tumor cell and targets it for death via an attached toxin. Because the toxins are typically of plant or bacterial origin, their clinical use is limited by immunogenicity and nonspecific toxicity. To circumvent these problems, we have begun to engineer immunotoxins containing human pancreatic ribonuclease. Here we describe the generation of ribonuclease mutants designed to evade a ubiquitous cytosolic inhibitor that would otherwise block cytotoxicity. Two mutants retained catalytic activity and were relatively resistant to the inhibitor. To deliver them to human T leukemic cells, these ribonuclease variants were fused to a single chain Fv fragment specific for CD 7. The ribonuclease-sFv fusion proteins bound CD 7(+) T cells and were internalized yet were not cytotoxic. Transfection of the proteins directly into the cytosol reduced cell viability, suggesting that the failure of the immunotoxins to kill cells when added externally resulted from the inability of the ribonuclease moiety to access the cytosol efficiently. Our results indicate appropriate intracellular routing, as well as resistance to inhibition, is critical to the cytotoxicity of human ribonuclease-based immunotoxins.

Role of aspartic acid 121 in human pancreatic ribonuclease catalysis

Bovine pancreatic ribonuclease (RNase A) is one of the most well studied enzymes of the ribonuclease family, unlike its human counterpart, the human pancreatic ribonuclease (HPR), whose physiological role in the body is not clearly understood. Human pancreatic ribonuclease consists of 128 amino acids and the main residues located in the active site of RNase A are also conserved in HPR. In the current study, to investigate the role of Asp-121 in the catalytic activity of human pancreatic ribonuclease, several variants were generated in which Asp-121 was either mutated to an alanine or C-terminal residues beyond Asp-121, and Phe-120 were deleted. The HPR mutants were cloned, expressed in E. coli and purified to homogeneity, and functionally characterized. The mutation D121A in HPR significantly decreased the rate of the enzymatic reaction, however this decrease was not universally observed for all substrates studied. Removal of the seven C-terminal amino acid residues thereby exposing Asp-121 yielded an HPR mutant with enhanced activity, however a further deletion removing Asp-121 resulted in the complete inactivation of HPR. Our results indicate that Asp-121 is crucial for the catalytic activity of HPR and may be involved in the depolymerization activity of the enzyme.

Human Ribonuclease A Superfamily Members, Eosinophil-Derived Neurotoxin and Pancreatic Ribonuclease, Induce Dendritic Cell Maturation and Activation

A number of mammalian antimicrobial proteins produced by neutrophils and cells of epithelial origin have chemotactic and activating effects on host cells, including cells of the immune system. Eosinophil granules contain an antimicrobial protein known as eosinophil-derived neurotoxin (EDN), which belongs to the RNase A superfamily. EDN has antiviral and chemotactic activities in vitro. In this study, we show that EDN, and to a lesser extent human pancreatic RNase (hPR), another RNase A superfamily member, activates human dendritic cells (DCs), leading to the production of a variety of inflammatory cytokines, chemokines, growth factors, and soluble receptors. Human angiogenin, a RNase evolutionarily more distant to EDN and hPR, did not display such activating effects. Additionally, EDN and hPR also induced phenotypic and functional maturation DCs. These RNases were as efficacious as TNF-alpha, but induced a different set of cytokine mediators. Furthermore, EDN production by human macrophages could be induced by proinflammatory stimuli. The results reveal the DC-activating activity of EDN and hPR and suggest that they are likely participants of inflammatory and immune responses. A number of endogenous mediators in addition to EDN have been reported to have both chemotactic and activating effects on APCs, and can thus amplify innate and Ag-specific immune responses to danger signals. We therefore propose these mediators be considered as endogenous multifunctional immune alarmins.

Cytosolic RNase Inhibitor Only Affects RNases with Intrinsic Cytotoxicity

Cytosolic RNase inhibitor binds to and neutralizes most members of the pancreatic type RNase superfamily. However, there are a few exceptions, e.g. amphibian onconase and bovine seminal RNase, and these are endowed with cytotoxic activity. Also, RNase variants created by mutagenesis to partially evade the RNase inhibitor acquire cytotoxic activity. These findings have led to the proposal that the cytosolic inhibitor acts as a sentry to protect mammalian cells from foreign RNases. We silenced the expression of the gene encoding the cytosolic inhibitor in HeLa cells and found that the cells become more sensitive to foreign cytotoxic RNases. However foreign, non-cytotoxic RNases remain non-cytotoxic. These results indicate that the cytosolic inhibitor neutralizes those foreign RNases that are intrinsically cytotoxic and have access to the cytosol. However, its normal physiological role may not be to guard against foreign RNases in general.

A fully human antitumor immunoRNase selective for ErbB-2-positive carcinomas.

We report the preparation and characterization of a novel, fully human antitumor immunoRNase (IR). The IR, a human RNase and fusion protein made up of a human single chain variable fragment (scFv), is directed to the ErbB-2 receptor and overexpressed in many carcinomas. The anti-ErbB-2 IR, named hERB-hRNase, retains the enzymatic activity of the wild-type enzyme (human pancreatic RNase) and specifically binds to ErbB-2-positive cells with the high affinity (K(d) = 4.5 nm) of the parental scFv. hERB-hRNase behaves as an immunoprotoxin and on internalization by target cells becomes selectively cytotoxic in a dose-dependent manner at nanomolar concentrations. Administered in five doses of 1.5 mg/kg to mice bearing an ErbB-2-positive tumor, hERB-hRNase induced a dramatic reduction in tumor volume. hERB-hRNase is the first fully human antitumor IR produced thus far, with a high potential as a poorly immunogenic human drug devoid of nonspecific toxicity, directed against ErbB-2-positive malignancies.

A Nuclear Localization Sequence Endows Human Pancreatic Ribonuclease with Cytotoxic Activity

Some members of the ribonuclease superfamily, such as Onconase, are cytotoxic to cancer cells. This is not the case for human pancreatic ribonuclease. This lack of cytotoxicity is probably a result of the inhibition exerted by the cytosolic ribonuclease inhibitor once the protein has reached the cytosol. Until now, all cytotoxic human pancreatic ribonuclease variants have been described as being resistant to the inhibitor. Here, we report on the characterization of a cytotoxic variant of human pancreatic ribonuclease which has an Arg triplet introduced onto one of its surface-exposed loops. Despite its sensitivity to the inhibitor, this variant, called PE5, was only 5-15 times less cytotoxic than Onconase. When it was taken up by cells, it was only observed within late compartments of the endocytic pathway, probably because the number of molecules transported to the cytosol was too small to allow their visualization. Nuclear import assays showed that the Arg triplet endows PE5 with a nuclear localization signal. In these experiments, PE5 was efficiently transported to the nucleus where it was initially localized in the nucleolus. Although the Arg introduction modified the net charge of the protein and somehow impaired recognition by the cytosolic inhibitor, control variants, which had the same number of charges or were not recognized by the inhibitor, were not toxic. We concluded that targeting a ribonuclease to the nucleus results in cytotoxicity. This effect is probably due to ribonuclease interference with rRNA processing and ribosome assembly within the nucleolus.

Selective abolition of pancreatic RNase binding to its inhibitor protein.

We have modified RNase inhibitor (RI) protein so that it no longer detectably binds pancreatic RNases but retains near-native affinity for human angiogenin (ANG). The K(i) value for RNase A is increased by a factor of >10(8), from 36 fM to >4 microM, and the selectivity factor for ANG is now >10(9). This dramatic change was achieved by remodeling the human RI loop segment Cys-408 -Leu-409 -Gly-410, which makes minor interactions with pancreatic RNase but does not contact ANG. The modifications selected were designed to sterically hinder docking of the undesired ligand. Three of the variants tested (C408W, G410W, and C408W/G410W) bind RNase A with almost the same avidity as WT RI. However, combination of the 408/410 double Trp replacement with deletion of the intervening residue, Leu-409, was sufficient to abolish inhibition of RNase A and human pancreatic RNase. The K(i) value for ANG with the deletion variant is 1.1 fM, only 2-fold higher than with WT RI. This variant may have potential utility both as an anticancer drug targeting ANG and as a tool for the investigation of the biological function of ANG. More generally, these findings demonstrate that a protein-protein interaction can be effectively and specifically disrupted by redesigning an interface region that makes no major energetic contribution to complex stability. This finding, in turn, may have implications for the development of small molecules that modulate protein-protein interactions.