<Emphasis Type="Italic">Renilla</Emphasis> luciferase-<Emphasis Type="Italic">Aequorea</Emphasis> GFP (Ruc-GFP) fusion protein,a novel dual reporter for real-time imaging of gene expression in cell cultures and in live animals

Light-emitting reporter proteins play an increasing role in the study of gene expression in vitro and in vivo. Here we present a ruc-gfp fusion gene construct generated by fusing a cDNA for Renilla luciferase (ruc) in-frame with a cDNA encoding the "humanized" GFP (gfp) from Aequorea. A plasmid containing the fusion gene construct was successfully transformed into, and expressed in, mammalian cells. The transformed cells exhibited both Renilla luciferase activity in the presence of coelenterazine and GFP fluorescence upon excitation with UV light. Spectrofluorometry of cells containing the Ruc-GFP fusion protein, in the absence of wavelengths capable of exciting GFP fluorescence but in the presence of the luciferase substrate, coelenterazine, showed an emission spectrum with two peaks at 475 nm and 508 nm. These two peaks correspond to the emission maximum of Renilla luciferase at 475 nm and that of GFP at 508 nm. The peak at 508 nm generated in the presence of coelenterazine alone (without UV excitation) is the result of intramolecular energy transfer from Renilla luciferase to Aequorea GFP. Southern analysis of genomic DNA purified from transformed Chinese hamster ovary (CHO) cells and fluorescence in situ hybridization (FISH) to metaphase chromosomes confirmed the integration of the ruc-gfp fusion gene on a single chromosome. The bifunctional Ruc-GFP fusion protein allows the detection of gene expression at the single-cell level based on green fluorescence, and in a group of cells based on luminescence emission. Furthermore, animal experiments revealed that light emission from the Ruc-GFP fusion protein can be detected externally in the organs or tissues of live animals bearing the gene construct.     

Detecting protein-protein interactions using Renilla luciferase fusion proteins

We have developed a novel system designated the luciferase assay for protein detection (LAPD) to study protein-protein interactions. This method involves two protein fusions, a soluble reporter fusion and a fusion for immobilizing the target protein. The soluble reporter is an N-terminal Renilla luciferase fusion protein that exhibits high Renilla luciferase activity. Crude cleared lysates from transfected Cos1 cells that express the Renilla luciferase fusion protein can be used in binding assays with immobilized target proteins. Following incubation and washing, target-bound Renilla luciferase fusion proteins produce light from the coelenterazine substrate, indicating an interaction between the two proteins of interest. As proof of the principle, we reproduced known, transient protein-protein interactions between the Cdc42 GTPase and its effector proteins. GTPase Renilla fusion proteins produced in Cos1 cells were tested with immobilized recombinant GST-N-WASP and CEP5 effector proteins. Using this assay, we could detect specific interactions of Cdc42 with these effector proteins in approximately 50 min. The specificity of these interactions was demonstrated by showing that they were GTPase-specific and GTP-dependent and not seen with other unrelated target proteins. These results suggest that the LAPD method, which is both rapid and sensitive, may have research and practical applications.     

Coelenterazine-binding protein of Renilla muelleri: cDNA cloning, overexpression, and characterization as a substrate of luciferase.

The Renilla bioluminescent system in vivo is comprised of three proteins--the luciferase, green-fluorescent protein, and coelenterazine-binding protein (CBP), previously called luciferin-binding protein (LBP). This work reports the cloning of the full-size cDNA encoding CBP from soft coral Renilla muelleri, its overexpression and properties of the recombinant protein. The apo-CBP was quantitatively converted to CBP by simple incubation with coelenterazine. The physicochemical properties of this recombinant CBP are determined to be practically the same as those reported for the CBP (LBP) of R. reniformis. CBP is a member of the four-EF-hand Ca(2+)-binding superfamily of proteins with only three of the EF-hand loops having the Ca(2+)-binding consensus sequences. There is weak sequence homology with the Ca(2+)-regulated photoproteins but only as a result of the necessary Ca(2+)-binding loop structure. In combination with Renilla luciferase, addition of only one Ca(2+) is sufficient to release the coelenterazine as a substrate for the luciferase for bioluminescence. This combination of the two proteins generates bioluminescence with higher reaction efficiency than using free coelenterazine alone as the substrate for luciferase. This increased quantum yield, a difference of bioluminescence spectra, and markedly different kinetics, implicate that a CBP-luciferase complex might be involved.     

A role for the insulin-like growth factor II/mannose-6-phosphate receptor in the insulin-induced inhibition of protein catabolism

Insulin and insulin-like growth factor (IGF)-I inhibit intracellular protein degradation in a variety of different cell types. In the present studies, the IGF-I-induced inhibition of protein metabolism in Chinese hamster ovary (CHO) cells was found to be blocked by polyclonal antibodies to the IGF-II/mannose-6-phosphate phosphate (Man-6-P) receptor, but not by control immunoglobulin. In contrast, these antibodies had no effect on the ability of IGF-I to stimulate glucose uptake in the same cells. The antibodies to the IGF-II/Man-6-P receptor also inhibited the effect of IGF-I and insulin on protein catabolism in human foreskin fibroblasts and human hepatoma cells, respectively. Moreover, CHO cells overexpressing a cDNA coding for the IGF-II/Man-6-P receptor were found to exhibit an increased effect of insulin on protein catabolism. In contrast, the insulin stimulation of glucose uptake is the same in these transfected cells as in the parental CHO cells. These results implicate the IGF-II/Man-6-P receptor in the insulin- and IGF-I-induced inhibition of protein catabolism.     

IGF binding protein-2 gene expression and the location of IGF-I and IGF-II in fetal rat lung.

Binding proteins for the insulin-like growth factors (IGF-BPs) are important modulators of the biological actions of IGF-I and IGF-II. The generation of IGFBPs within developing organs, and their spatial arrangement, may similarly determine IGF action at specific microanatomical sites. In situ hybridization studies with late gestation (days 16, 18 and 20) fetal rat lung using a cDNA probe for IGFBP-2 showed strong gene expression in the fetal lung epithelial structures (alveoli and airways). The sites of IGFBP-2 gene expression were associated with immunoreactive IGF-II at the apical surface of the epithelium. By day 20, there was also some IGFBP-2 gene expression and immunoreactive IGF-II at discrete sites in the mesenchyme. In contrast, immunoreactive IGF-I was found predominantly distributed in a punctate pattern, consistent with its presence in the lumen or walls of small vessels or capillaries, and in a granular, intracellular form in both epithelial and mesenchymal cells. These studies suggest that endogenously generated IGFBP-2 may determine the distribution of IGF-II, principally at the apical surface of lung epithelia. IGF-I does not colocalise with IGF-II peptide or the sites of IGFBP-2 gene expression. We conclude that the spatial distributions of these two related growth factors are separately controlled, to some extent by endogenously generated binding proteins.     

Expression, purification and characterization of calcium-triggered luciferin-binding protein of Renilla reniformis

The Ca2+-triggered luciferin-binding protein of Renilla reniformis (RLBP) is a non-covalent complex of apoprotein (apoRLBP) and coelenterazine (luciferin). The gene encoding apoRLBP with 552 nucleotides has been synthesized by assembly PCR methods with synthetic oligonucleotides, and the histidine-tagged apoRLBP expressed as a soluble form in the periplasmic space of Escherichia coli cells. The apoRLBP was purified by nickel chelate chromatography and the procedure yielded 18.2mg of recombinant apoRLBP from 80 ml of cultured cells with purity greater than 95%. The purified apoRLBP was converted to RLBP by incubation with coelenterazine in the presence of dithiothreitol and the purity of recombinant RLBP was estimated to be over 95% by comparison with the absorption spectral data of native RLBP. When RLBP mixed with Ca2+, coelenterazine was dissociated from RLBP and was utilized for the luminescence reaction of Renilla luciferase. Also semi-synthetic RLBPs with h-, e-, and Bis-coelenterazines were prepared and characterized.     

Structure based mechanism of the Ca(2+)-induced release of coelenterazine from the Renilla binding protein

The crystal structure of the Ca(2+)-loaded coelenterazine-binding protein from Renilla muelleri in its apo-state has been determined at resolution 1.8 A. Although calcium binding hardly affects the compact scaffold and overall fold of the structure before calcium addition, there are easily discerned shifts in the residues that were interacting with the coelenterazine and a repositioning of helices, to expose a cavity to the external solvent. Altogether these changes offer a straightforward explanation for how following the addition of Ca(2+), the coelenterazine could escape and become available for bioluminescence on Renilla luciferase. A docking computation supports the possibility of a luciferase-binding protein complex.     

Functional complementation of high-efficiency resonance energy transfer: a new tool for the study of protein binding interactions in living cells

Green bioluminescence in Renilla species is generated by a approximately 100% efficient RET (resonance energy transfer) process that is caused by the direct association of a blue-emitting luciferase [Rluc (Renilla luciferase)] and an RGFP (Renilla green fluorescent protein). Despite the high efficiency, such a system has never been evaluated as a potential reporter of protein-protein interactions. To address the question, we compared and analysed in mammalian cells the bioluminescence of Rluc and RGFP co-expressed as free native proteins, or as fused single-chain polypeptides and tethered partners of self-assembling coiled coils. Here, we show that: (i) no spontaneous interactions generating detectable BRET (bioluminescence RET) signals occur between the free native proteins; (ii) high-efficiency BRET similar to that observed in Renilla occurs in both fusion proteins and self-interacting chimaeras, but only if the N-terminal of RGFP is free; (iii) the high-efficiency BRET interaction is associated with a dramatic increase in light output when the luminescent reaction is triggered by low-quantum yield coelenterazine analogues. Here, we propose a new functional complementation assay based on the detection of the high-efficiency BRET signal that is generated when the reporters Rluc and RGFP are brought into close proximity by a pair of interacting proteins to which they are linked. To demonstrate its performance, we implemented the assay to measure the interaction between GPCRs (G-protein-coupled receptors) and beta-arrestins. We show that complementation-induced BRET allows detection of the GPCR-beta-arrestin interaction in a simple luminometric assay with high signal-to-noise ratio, good dynamic range and rapid response.     

IGF-II and IGFBP-6 regulate cellular contractility and proliferation in Dupuytren's disease

Dupuytren's disease (DD) is a common and heritable fibrosis of the palmar fascia that typically manifests as permanent finger contractures. The molecular interactions that induce the development of hyper-contractile fibroblasts, or myofibroblasts, in DD are poorly understood. We have identified IGF2 and IGFBP6, encoding insulin-like growth factor (IGF)-II and IGF binding protein (IGFBP)-6 respectively, as reciprocally dysregulated genes and proteins in primary cells derived from contracture tissues (DD cells). Recombinant IGFBP-6 inhibited the proliferation of DD cells, patient-matched control (PF) cells and normal palmar fascia (CT) cells. Co-treatments with IGF-II, a high affinity IGFBP-6 ligand, were unable to rescue these effects. A non-IGF-II binding analog of IGFBP-6 also inhibited cellular proliferation, implicating IGF-II-independent roles for IGFBP-6 in this process. IGF-II enhanced the proliferation of CT cells, but not DD or PF cells, and significantly enhanced DD and PF cell contractility in stressed collagen lattices. While IGFBP-6 treatment did not affect cellular contractility, it abrogated the IGF-II-induced contractility of DD and PF cells in stressed collagen lattices. IGF-II also significantly increased the contraction of DD cells in relaxed lattices, however this effect was not evident in relaxed collagen lattices containing PF cells. The disparate effects of IGF-II on DD and PF cells in relaxed and stressed contraction models suggest that IGF-II can enhance lattice contractility through more than one mechanism. This is the first report to implicate IGFBP-6 as a suppressor of cellular proliferation and IGF-II as an inducer of cellular contractility in this connective tissue disease.     

High-level expression of human insulin-like growth factor II in Escherichia coli.

A gene encoding mature human insulin-like growth factor II (IGF-II) was constructed from the modified IGF-II cDNA sequence and two double-stranded synthetic oligodeoxynucleotide linkers. It was fused to a truncated lacZ gene such that IGF-II was expressed as part of C-terminus of beta-galactosidase. This fused lacZ'-IGF-II gene was under the control of tac promoter and we overproduced the beta-galactosidase-IGF-II fusion protein in the Escherichia coli. The fusion protein formed inclusion bodies inside the cells. The fusion protein was purified from the isolated inclusion bodies and IGF-II protein was obtained from their fusion protein by CNBr cleavage. The released IGF-II was confirmed by its molecular weight as determined by SDS-PAGE and by its ability to bind anti-IGF antibody.     

Purification and characterization of native human insulin-like growth factor binding protein-6

Insulin-like growth factor binding proteins (IGFBPs) are key regulators of insulin-like growth factor (IGF) mediated signal transduction and thereby can profoundly influence cellular phenotypes and cell fate. Whereas IGFBPs are extracellular proteins, intracellular activities were described for several IGFBP family members, such as IGFBP-3, which can be reinternalized by endocytosis and reaches the nucleus through routes that remain to be fully established. Within the family of IGFBPs, IGFBP-6 is unique for its specific binding to IGF-II. IGFBP-6 was described to possess additional IGF-independent activities, which have in part been attributed to its translocation to the nucleus; however, cellular uptake of IGFBP-6 was not described. To further explore IGFBP-6 functions, we developed a new method for the purification of native human IGFBP-6 from cell culture supernatants, involving a four-step affinity purification procedure, which yields highly enriched IGFBP-6. Whereas protein purified in this way retained the capacity to interact with IGF-II and modulate IGF-dependent signal transduction, our data suggest that, unlike IGFBP-3, human IGFBP-6 is not readily internalized by human tumor cells. To summarize, this work describes a novel and efficient method for the purification of native human insulin-like growth factor binding protein 6 (IGFBP-6) from human cell culture supernatants, applying a four-step chromatography procedure. Intactness of purified IGFBP-6 was confirmed by IGF ligand Western blot and ability to modulate IGF-dependent signal transduction. Cellular uptake studies were performed to further characterize the purified protein, showing no short-term uptake of IGFBP-6, in contrast to IGFBP-3.     

Alteration in pancreatic immunoreactivity of insulin-like growth factor (IGF)-binding protein (IGFBP)-6 and in intracellular degradation of IGFBP-3 in fibroblasts of IGF-II receptor/IGF-II-deficient mice.

The Type-2 insulin-like growth factor receptor (IGF2R) mediates the transport of lysosomal hydrolases to lysosomes and the clearance of insulin-like growth factor II (IGF-II). Mutant mice lacking IGF2R usually die perinatally, but are completely rescued from lethality in the absence of IGF-II. IGF2R/IGF-II-deficient mice have elevated levels of circulating IGF binding protein (IGFBP)-3 and show a strong IGFBP-6 immunoreactivity in all pancreatic islet cells and in secretory granules of different size in acinar cells and interlobular connective tissue of exocrine pancreas. Fibroblasts derived from double mutant mice missort the lysosomal protease cathepsin D, and are able to degrade endocytosed (125I)IGFBP-3 intracellularly, however, with lower efficiency than in control cells. These results show that the deficiency of IGF2R and IGF-II affects the expression and metabolism of IGFBPs in a tissue- and cell type-specific manner.     

Crystal structures of the luciferase and green fluorescent protein from Renilla reniformis

Due to its ability to emit light, the luciferase from Renilla reniformis (RLuc) is widely employed in molecular biology as a reporter gene in cell culture experiments and small animal imaging. To accomplish this bioluminescence, the 37-kDa enzyme catalyzes the degradation of its substrate coelenterazine in the presence of molecular oxygen, resulting in the product coelenteramide, carbon dioxide, and the desired photon of light. We successfully crystallized a stabilized variant of this important protein (RLuc8) and herein present the first structures for any coelenterazine-using luciferase. These structures are based on high-resolution data measured to 1.4 Å and demonstrate a classic α/β-hydrolase fold. We also present data of a coelenteramide-bound luciferase and reason that this structure represents a secondary conformational form following shift of the product out of the primary active site. During the course of this work, the structure of the luciferase's accessory green fluorescent protein (RrGFP) was also determined and shown to be highly similar to that of Aequorea victoria GFP.     

Insulin-like growth factor family and combined antisense approach in therapy of lung carcinoma

BACKGROUND: Perturbation in a level of any peptide from insulin-like growth factor (IGF) family (ligands, receptors, and binding proteins) seems to be implicated in lung cancer formation; IGF ligands and IGF-I receptor through their mitogenic and anti-apoptotic action, and the mannose 6-phosphate/insulin-like growth factor II receptor (M6-P/IGF-IIR) possibly as a tumor suppressor. MATERIALS AND METHODS: To determine the identity, role, and mutual relationship of IGFs in lung cancer growth and maintenance, we examined IGF's gene (by RT-PCR) and protein (by immunohistochemistry) expression in 69 human lung carcinoma tissues. We also examined IGF-I receptor numbers (Scatchard analysis) and IGF-II production and release (by Western blot) in IGF-II/IGF-IR mRNA positive and negative lung carcinomas. Finally, the potential role of IGF-IR and IGF-II as growth promoting factors in lung cancer was studied using antisense oligodeoxynucleotides that specifically inhibit IGF-IR and IGF-II mRNA. RESULTS: Thirty-two tumors were positive for IGF-I, 39 for IGF-II, 48 for IGF-IR, and 35 for IGFBP-4 mRNA. Seventeen tumors were concomitantly positive for all four IGFs, whereas 34 were positive for IGF-II, IGF-IR, and IGFBP-4 mRNA. An elevated amount of IGF-II peptide was secreted into the growth medium of cell cultures established from five different IGF-II/IGF-IR mRNA positive lung cancer tissues. The cells also expressed elevated numbers of IGF-IR. Nine IGF-II-negative and 19 IGF-II-positive lung cancers of different stages were selected, and M6-P/ IGF-II receptor was determined immunohistochemically. Most of the IGF-II-negative tumors were strongly positive for M6-P/IGF-IIR. IGF-II-positive tumors were mostly negative for M6-P/IGF-II receptors. Antisense oligodeoxynucleotides to IGF-II significantly inhibited, by 25-60%, the in vitro growth of all six lung cancer cell lines. However, the best results (growth inhibition of up to 80%) were achieved with concomitant antisense treatment (to IGF-IR and IGF-II). CONCLUSION: Our data suggest that lung cancer cells produce IGF-IR and IGF-II, which in turn stimulates their proliferation by autocrine mechanism. Cancer cell proliferation can be abrogated or alleviated by blocking the mRNA activity of these genes indicating that an antisense approach may represent an effective and practical cancer gene therapy strategy.     

Expression, purification, and in vitro characterization of recombinant salmon insulin-like growth factor-II

The insulin-like growth factors, IGF-I and IGF-II, are single chain polypeptides, which are structurally related to proinsulin and promote proliferation and differentiation of cells in many vertebrate species. Previous attempts to produce recombinant salmon IGF-II (rsIGF-II) were compromised by low expression levels and co-purification of incorrectly cleaved protein with the authentic recombinant product. In this study, a gene containing the coding region for Atlantic salmon (Salmo salar) IGF-II was cloned into a modified pET32a expression vector and transformed into Escherichia coli BL21 trxB (DE3) cells. Upon growth and induction (with IPTG) of the transformant, recombinant salmon IGF-II (rsIGF-II) was expressed as an insoluble, 28kDa thioredoxin.sIGF-II fusion protein linked by a protease cleavage motif (trx.FAHY.sIGF-II) in inclusion bodies. The inclusion bodies were subsequently solubilized and the fusion protein was purified by Ni-affinity chromatography. Recombinant IGF-II (7.8kDa) was then released from the fusion partner using H64A subtilisin BPN' protease and purified by reversed-phase HPLC. Homogeneity of the final recombinant product was confirmed by N-terminal amino acid sequencing, ion-spray mass spectrometry, SDS-polyacrylamide gel electrophoresis, and analytical reversed-phase HPLC. The biological activity of rsIGF-II was demonstrated in cultured rat L6 myoblasts and was found to be approximately 9- and 5-fold less potent than recombinant human IGF-I and recombinant salmon IGF-I, respectively, a result similar to that demonstrated previously with other recombinant fish IGF-II's in non-homologous cell lines.     

In vivo testing of Renilla luciferase substrate analogs in an orthotopic murine model of human glioblastoma

In vivo bioluminescent imaging using cells expressing Renilla luciferase is becoming increasingly common. Hindrances to the more widespread use of Renilla luciferase are the high autoluminescence of its natural substrate, coelenterazine, in plasma, the relatively high absorbance by tissue of the light emitted by the enzyme-substrate reaction; rapid clearance of the substrate; and significant cost. These factors, save for the cost, which has its own limiting effect on use, can combine to reduce the sensitivity of in vivo assays utilizing this reporter system, and methods of increasing light output or decreasing autoluminescence could be of great benefit. A number of analogs of coelenterazine are being investigated may accomplish one or both of these goals. In this study that we report on the testing of two new substrate analogs, EnduRen and ViViren, manufactured by Promega Corporation, in an orthotopic murine model of human glioblastoma expressing Renilla luciferase. We have tested these analogs in this cell line both in vitro and in vivo, and find that the substrate viviren results in significantly greater light output than the natural substrate or the other analog EnduRen. This new substrate could be valuable for studies where greater sensitivity is important.     

Interaction of insulin-like growth factor II (IGF-II) with multiple plasma proteins: high affinity binding of plasminogen to IGF-II and IGF-binding protein-3

In the circulation, most of the insulin-like growth factors (IGFs), IGF-binding proteins (IGFBPs), and IGFBP proteases are bound in high molecular mass complexes of > or =150 kDa. To investigate molecular interactions between proteins involved in IGF.IGFBP complexes, Cohn fraction IV of human plasma was subjected to IGF-II affinity chromatography followed by reversed-phase high pressure liquid chromatography and analysis of bound proteins. Mass spectrometry and Western blotting revealed the presence of IGFBP-3, IGFBP-5, transferrin, plasminogen, prekallikrein, antithrombin III, and the soluble IGF-II/mannose 6-phosphate receptor in the eluate. Furthermore, an IGFBP-3 protease cleaving also IGFBP-2 but not IGFBP-4 was co-purified from the IGF-II column. Inhibitor studies and IGFBP-3 zymography have demonstrated that the 92-kDa IGFBP-3 protease belongs to the class of serine-dependent proteases. IGF-II ligand blotting and surface plasmon resonance spectrometry have been used to identify plasminogen as a novel high affinity IGF-II-binding protein capable of binding to IGFBP-3 with 50-fold higher affinity than transferrin. In combination with transferrin, the overall binding constant of plasminogen/transferrin for IGF-II was reduced 7-fold. Size exclusion chromatography of the IGF-II matrix eluate revealed that transferrin, plasminogen, and the IGFBP-3 protease are present in different high molecular mass complexes of > or =440 kDa. The present data indicate that IGFs, low and high affinity IGFBPs, several IGFBP-associated proteins, and IGFBP proteases can interact, which may result in the formation of binary, ternary, and higher molecular weight complexes capable of modulating IGF binding properties and the stability of IGFBPs.