Mammalian Cell Expression System Research Articles

Enhanced Glycosylation Detection and Characterization of a Human IgG1 Reference Material by using 2D‐LC MS/MS Fractionation and High Resolution Mass Spectral Library

IntroductionThe structure analysis of glycans remains challenging due to the lack of high‐throughput analytical platforms and freely available automated software capable of analyzing spectral data and determine the compositions and structures of glycopeptides and glycans. Here we describe the in‐depth characterization and relative quantification of N‐glycosylation in an IgG1 reference material (NISTmAb) and commercial mAbs using liquid chromatography and tandem mass spectrometer (1D/2D LC‐MS/MS) and the mass spectral library.MethodsDetailed analyses were carried out of glycopeptides produced in the tryptic digestion of the NISTmAb and other commercial mAbs. The method includes (1) nanospray ESI LC–MS/MS over a wide range of HCD collision energies with both conventional 1D separation for various digestion conditions and a 20/24 fraction 2D‐LC study of a single digest; (2) energy‐dependent changes in HCD fragmentation to determine the proposed glycan structures over a wide range of NCE values: 12, 16, 20, 24, 28, 32, 36, and 40; (3) the development of an automated glycopeptide analysis software tool; and (4) the creation of a publicly available high resolution spectral library of glycopeptides. This resulted in 800 1D and 300 2D runs and a glycopeptide spectral library comprising 80 glycans with different masses, and around 2000 HCD spectra of glycopeptides obtained in IgG1 mAbs expressed in NS0 and CHO cell lines.ResultsDerived from 195 different glycopeptides found in 1D and 247 glycopeptides found in 2D runs, more than 60 unique glycan structures in the NISTmAb were confidently determined. This is a significantly larger number of glycans than found in any other glycoanalysis of therapeutic glycoproteins, exhibiting a greater variety of low‐abundance glycan structures detected in 2D studies compared to that of 1D and released glycan studies. This comparison directly illustrates the added depth of the 2D studies leading to the extended abundance range and glycoanalysis coverage. To summarize, the IgG‐Fc N‐linked glycosylation profile of NISTmAb was predominated by asialylated, biantennary core fucosylated complex glycans, which are well‐known main glycoforms observed in IgG1 mAbs produced by all major mammalian cell expression systems. However, significantly higher amounts of α‐linked galactosylation and sialylation were also observed in the 2D study. This profile revealed major differences between the cell lines in the glycoforms produced: the NS0 cell line used for making the IgG1 under study expressed glycoforms with higher extents of nonhuman α‐Gal and Neu5GC residues. In addition, an extensive analysis of HCD glycopeptide fragmentation at multiple low HCD energies confirmed the newly proposed glycan compositions, in‐source glycan fragments, glycan rearrangement products, multiple structural isomers, and glycan modifications including sulfated N‐glycans. This led to the creation of a first high‐resolution, peak‐annotated mass spectral library to aid the analysis of glycopeptides derived from IgG1 drugs. This library is freely available online at http://chemdata.nist.gov/dokuwiki/doku.php?id=peptidew:lib:newigghsalib along with search software.Support or Funding InformationThis work was supported solely with NIST funds. Certain commercial equipment, instruments, or materials are identified in this presentation to specify the experimental procedure adequately. Such an identification is not intended to imply recommendation or endorsement by the National Institute of Standards and Technology, nor is it intended to imply that the materials or equipment identified are necessarily the best available for the purpose.

Light-Responsive Promoters.

Recent advances in the development of light-inducible transgene expression systems have overcome many inherent drawbacks of conventional chemically regulated systems. The latest generation of those light-regulated systems that are specifically responsive to different wavelengths allows spatiotemporal control of gene expression in a so far unprecedented manner.In this chapter, we first describe the available light-inducible gene expression systems compatible with mammalian cells and explain their underlying mechanisms. Afterward, we give a detailed protocol for the implementation of a UVB light-inducible expression system in mammalian cells.

Efficient Production of a Bioactive Bevacizumab Monoclonal Antibody Using the 2A Self-cleavage Peptide in Transgenic Rice Callus.

Bevacizumab, a humanized monoclonal antibody (mAb) targeting to the vascular endothelial growth factor (VEGF), has been widely used in clinical practice for the treatment of multiple cancers. Bevacizumab was mostly produced by the mammalian cell expression system. We here reported the first plant-derived Bevacizumab by using transgenic rice callus as an alternative gene expression system. Codon-optimized Bevacizumab light chain (BLC) and Bevacizumab heavy chain (BHC) genes were designed, synthesized as a polyprotein with a 2A self-cleavage linker peptide from the Foot-and-mouth disease virus, cloned into a plant binary vector under a constitutive maize ubiquitin promoter, and transformed into rice nuclear genome through Agrobacterium-mediated transformation. Southern blot and western blot analyses confirmed the integration and expression of BLC and BHC genes in transgenic rice callus. Enzyme-linked immunosorbent assay (ELISA) analysis indicated that the rice-derived Bevacizumab mAb was biologically active and the recombinant mAb was expressed at high levels (160.7–242.8 mg/Kg) in transgenic rice callus. The mAb was purified by using protein A affinity chromatography and the purified antibody was tested for its binding affinity with its target human VEGF (hVEGF) antigen by ELISA. Rice callus produced Bevacizumab and a commercial Bevacizumab (Avastin) were shown to have similar binding affinity to hVEGF. These results indicated that rice callus produced Bevacizumab could have similar biological activity and might potentially be used as a cost-effective biosimilar molecule in future cancer treatment.

Hepatitis C virus: The role of N-glycosylation sites of viral genotype 1b proteins for formation of viral particles in insect and mammalian cells

Hepatitis C virus (HCV) is characterized by considerable genetic variability and, as a consequence, it has 6 genotypes and multitude of subtypes. HCV envelope glycoproteins are involved in the virion formation; the correct folding of these proteins plays the key role in virus infectivity. Glycosylation at certain sites of different genotypes HCV glycoproteins shows substantial differences in functions of the individual glycans (Goffard et al., 2005; Helle et al., 2010) [1,2]. In this study, differential glycosylation sites of HCV genotype 1b envelope proteins in insect and mammalian cells was demonstrated. We showed that part of glycosylation sites was important for folding of the proteins involved in the formation of viral particles. Point mutations were introduced in the protein N-glycosylation sites of HCV (genotype 1b) and the mutant proteins were analyzed using baculovirus expression system in mammalian and insect cells. Our data showed that, in contrast to HCV 1a and 2a, the folding of HCV 1b envelope proteins E2 (sites N1, N2, N10) and E1 (sites N1, N5) was disrupted, however that did not prevent the formation of virus-like particles (VLP) with misfolded glycoproteins having densities typical for HCV particles containing RNA fragments. Experimental data are supported by mathematical modeling of the structure of E1 mutant variants.

1C22 内耳タンパク質モーターprestinの哺乳類細胞発現系の構築

1C22 内耳タンパク質モーターprestinの哺乳類細胞発現系の構築

Abstract 348: Amino acid-based prodrugs of gemcitabine - a therapeutic option to overcome chemoresistance in pancreatic cancer

Abstract Pancreatic cancer is the deadliest of all cancers. Currently, gemcitabine is the only drug of choice to treat this cancer but the results are modest as the disease itself is diagnosed at an advanced stage and often times develops resistance to gemcitabine via silencing of the influx transporters for the drug. We hypothesized that the delivery of gemcitabine into pancreatic cancer cells in a different form via some other transporters would provide a logical alternative approach. It would even be better if we can exploit for this purpose the transporters that are selectively upregulated in pancreatic cancer. Several nucleoside drugs are transportable substrates for the amino acid transporter SLC6A14 if used in the form of amino acid-based prodrugs. Such strategy would work for gemcitabine if SLC6A14 is upregulated in pancreatic cancer. With this in mind, we analyzed publically available microarray datasets for the amino acid transporters that are upregulated in pancreatic cancer. We found four transporters (SLC1A5, SLC7A5, SLC7A11, and SLC6A14) overexpressed in tumors compared to normal tissue. The fold-increase in expression is the highest for SLC6A14 in all datasets (range, 1.3 - 163.3; p, 0.05 - 2×10−14). The fold-increase for the other three transporters is 2 at the maximum. We confirmed these findings using primary pancreatic cancer tissues and cancer cell lines. First, we used pancreatic cancer tissues and compared with the normal tissues. SLC6A14 mRNA is increased at least by 7-fold in tumors versus normal tissue. We then evaluated the expression of SLC6A14 mRNA and protein in normal and pancreatic cancer cell lines. The expression is higher in all cancer cell lines than in a normal cell line. This was further confirmed with a tissue microarray containing pancreatic tumor tissues from 52 patients. The delivery of gemcitabine in the form of amino acid-based prodrugs via SLC6A14, an exceptionally energy-coupled transporter, as a means to overcome gemcitabine resistance is a novel idea that has never been tested. As a proof of principle, we have shown that SLC6A14 can transport several nucleoside drugs (acyclovir, ganciclovir, and cytarabine) in the form of amino acid-based prodrugs. The parent nucleosides are not substrates for the transporter. To specifically assess the interaction of amino acid-based prodrugs of gemcitabine with SLC6A14, we synthesized the valyl ester of gemcitabine and investigated its transport via SLC6A14 using two different heterologous expression systems: a mammalian cell expression system and the Xenopus oocyte expression system. In both experimental approaches, we found that the valyl ester of gemcitabine, but not the parent drug, is a transportable substrate for SLC6A14. The transport process is Na/Cl-coupled and electrogenic. We conclude that amino acid-based prodrugs of gemcitabine represent a viable alternative to overcome resistance to gemcitabine in the treatment of pancreatic cancer. Citation Format: Yangzom D. Bhutia, Ellappan Babu, Pankaj K. Singh, Vadivel Ganapathy. Amino acid-based prodrugs of gemcitabine - a therapeutic option to overcome chemoresistance in pancreatic cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 348. doi:10.1158/1538-7445.AM2015-348

The production of KIR–Fc fusion proteins and their use in a multiplex HLA class I binding assay

Soluble recombinant proteins that comprise the extracellular part of a surface expressed receptor attached to the Fc region of an IgG antibody have facilitated the determination of ligand specificity for an array of immune system receptors. Among such receptors is the family of killer cell immunoglobulin-like receptors (KIR) that recognize HLA class I ligands. These receptors, expressed on natural killer (NK) cells and T cells, play important roles in both immune defense and placental development in early pregnancy. Here we describe a method for the production of two domain KIR–Fc fusion proteins using baculovirus infected insect cells. This method is more scalable than traditional mammalian cell expression systems and produces efficiently folded proteins that carry posttranslational modifications found in native KIR. We also describe a multiplex binding assay using the Luminex platform that determines the avidity and specificity of two domain KIR–Fc for a panel of microbeads, each coated with one of 97 HLA class I allotypes. This assay is simple to perform, and represents a major improvement over the assays used previously, which were limited in the number of KIR and HLA class I combinations that could be assayed at any one time. The results obtained from this assay can be used to predict the response of NK cell and T cells when their KIR recognize HLA class I.

Role of cytochrome P450 2C8*3 (CYP2C8*3) in paclitaxel metabolism and paclitaxel-induced neurotoxicity.

The CYP2C8*3 allele has been suggested as a risk factor for paclitaxel-induced neuropathy but the data hitherto published are conflicting. In total 435 patients were investigated with respect to maximum neuropathy grade and accumulated paclitaxel dose. The enzymatic properties of CYP2C8.3 variant were analyzed using heterologous mammalian HEK293 cell expression system. No significant association between CYP2C8*3 allele and neuropathy was found, although a trend was observed. The paclitaxel and amodiaquine metabolism by CYP2C8.3 were found similar to CYP2C8.1, whereas CYP2C8.3 was more efficient in the metabolism of rosiglitazone. These results indicate a difference in substrate specificity between CYP2C8.1 and CYP2C8.3; however, the CYP2C8*3 allele has no major impact on paclitaxel metabolism in vitro or of paclitaxel-induced neuropathy in vivo. Original submitted on 6 February 2015; revision submitted on 9 April 2015.

Structural Constraints Determine the Glycosylation of HIV-1 Envelope Trimers.

A highly glycosylated, trimeric envelope glycoprotein (Env) mediates HIV-1 cell entry. The high density and heterogeneity of the glycans shield Env from recognition by the immune system, but paradoxically, many potent broadly neutralizing antibodies (bNAbs) recognize epitopes involving this glycan shield. To better understand Env glycosylation and its role in bNAb recognition, we characterized a soluble, cleaved recombinant trimer (BG505 SOSIP.664) that is a close structural and antigenic mimic of native Env. Large, unprocessed oligomannose-type structures (Man8-9GlcNAc2) are notably prevalent on the gp120 components of the trimer, irrespective of the mammalian cell expression system or the bNAb used for affinity purification. In contrast, gp41 subunits carry more highly processed glycans. The glycans on uncleaved, non-native oligomeric gp140 proteins are also highly processed. A homogeneous, oligomannose-dominated glycan profile is therefore a hallmark of a native Env conformation and a potential Achilles' heel that can be exploited for bNAb recognition and vaccine design.

Versatility of Homogeneous Time-Resolved Fluorescence Resonance Energy Transfer Assays for Biologics Drug Discovery

Identification of potential lead antibodies in the drug discovery process requires the use of assays that not only measure binding of the antibody to the target molecule but assess a wide range of other characteristics. These include affinity ranking, measurement of their ability to inhibit relevant protein-protein interactions, assessment of their selectivity for the target protein, and determination of their species cross-reactivity profiles to support in vivo studies. Time-resolved fluorescence resonance energy transfer is a technology that offers the flexibility for development of such assays, through the availability of donor and acceptor fluorophore-conjugated reagents for detection of multiple tags or fusion proteins. The time-resolved component of the technology reduces potential assay interference, allowing screening of a range of different crude sample types derived from the bacterial or mammalian cell expression systems often used for antibody discovery projects. Here we describe the successful application of this technology across multiple projects targeting soluble proteins and demonstrate how it has provided key information for the isolation of potential therapeutic antibodies with the desired activity profile.

A stable human-cell system overexpressing cystic fibrosis transmembrane conductance regulator recombinant protein at the cell surface.

Recent human clinical trials results demonstrated successful treatment for certain genetic forms of cystic fibrosis (CF). To extend treatment opportunities to those afflicted with other genetic forms of CF disease, structural and biophysical characterization of CF transmembrane conductance regulator (CFTR) is urgently needed. In this study, CFTR was modified with various tags, including a His10 purification tag, the SUMOstar (SUMO*) domain, an extracellular FLAG epitope, and an enhanced green fluorescent protein (EGFP), each alone or in various combinations. Expressed in HEK293 cells, recombinant CFTR proteins underwent complex glycosylation, compartmentalized with the plasma membrane, and exhibited regulated chloride-channel activity with only modest alterations in channel conductance and gating kinetics. Surface CFTR expression level was enhanced by the presence of SUMO* on the N-terminus. Quantitative mass-spectrometric analysis indicated approximately 10% of the total recombinant CFTR (SUMO*-CFTR(FLAG)-EGFP) localized to the plasma membrane. Trial purification using dodecylmaltoside for membrane protein extraction reproducibly recovered 178 ± 56 μg SUMO*-CFTR(FLAG)-EGFP per billion cells at 80% purity. Fluorescence size-exclusion chromatography indicated purified CFTR was monodisperse. These findings demonstrate a stable mammalian cell expression system capable of producing human CFTR of sufficient quality and quantity to augment future CF drug discovery efforts, including biophysical and structural studies.

Strategies for recombinant production of human glycosylation enzymes: Comparison of mammalian and insect cell expression systems

Strategies for recombinant production of human glycosylation enzymes: Comparison of mammalian and insect cell expression systems

Recombinant protein expression for structural biology in HEK 293F suspension cells: a novel and accessible approach.

The expression and purification of large amounts of recombinant protein complexes is an essential requirement for structural biology studies. For over two decades, prokaryotic expression systems such as E. coli have dominated the scientific literature over costly and less efficient eukaryotic cell lines. Despite the clear advantage in terms of yields and costs of expressing recombinant proteins in bacteria, the absence of specific co-factors, chaperones and post-translational modifications may cause loss of function, mis-folding and can disrupt protein-protein interactions of certain eukaryotic multi-subunit complexes, surface receptors and secreted proteins. The use of mammalian cell expression systems can address these drawbacks since they provide a eukaryotic expression environment. However, low protein yields and high costs of such methods have until recently limited their use for structural biology. Here we describe a simple and accessible method for expressing and purifying milligram quantities of protein by performing transient transfections of suspension grown HEK (Human Embryonic Kidney) 293F cells.

Recombinant Protein Expression for Structural Biology in HEK 293F Suspension Cells: A Novel and Accessible Approach

The expression and purification of large amounts of recombinant protein complexes is an essential requirement for structural biology studies. For over two decades, prokaryotic expression systems such as E. coli have dominated the scientific literature over costly and less efficient eukaryotic cell lines. Despite the clear advantage in terms of yields and costs of expressing recombinant proteins in bacteria, the absence of specific co-factors, chaperones and post-translational modifications may cause loss of function, mis-folding and can disrupt protein-protein interactions of certain eukaryotic multi-subunit complexes, surface receptors and secreted proteins. The use of mammalian cell expression systems can address these drawbacks since they provide a eukaryotic expression environment. However, low protein yields and high costs of such methods have until recently limited their use for structural biology. Here we describe a simple and accessible method for expressing and purifying milligram quantities of protein by performing transient transfections of suspension grown HEK (Human Embryonic Kidney) 293F cells.

Recombinant human BMPs 2 and 4 expressed in mammalian cells aiming at bone tissue engineering and stem cell proliferation and differentiation

Background Bone Morphogenetic Proteins (BMPs) are multifunctional, secreted cytokines belonging to the TGF-b superfamily. These proteins act as a disulfide-linked homodimer, being potent regulators of bone and cartilage formation and repair, cell proliferation in embryonic development and adult bone homeostasis. BMPs are promising molecules in periodontal regeneration to treat physiopathological bone loss and non-union fractures and in oral surgery, and to accelerate and increase osseointegration. BMPs are dimeric molecules displaying sites for Nand O-glycosylation, which increases the stability and half-life of the protein in the body, in addition to determining the specificity of receptor coupling. BMP-2 induces cartilage and bone formation. BMP4 has also been shown to play a role in triggering osteoblastic differentiation of mesenchymal stem cells, through activation of osteoblastic related genes. In order to ensure proper glycosylation and conformational folding and to prevent immunogenicity, we elected a mammalian cell expression system to produce these BMPs aiming at bone regeneration, stem cell proliferation and differentiation and their application in human and veterinary cell therapy.

Rapid production of HIV-1 neutralizing antibodies in baculovirus infected insect cells

Broadly neutralizing antibodies have been shown promise as prophylactic and therapeutic agents to provide passive protection against human immunodeficiency virus type 1 (HIV-1) infection. Such protein based microbicides are traditionally produced using mammalian cell expression system, which is not able to satisfy the increasing demand of these proteins in large scale. In this report, two of HIV-1 broadly neutralizing antibodies, b12 and VRC01, were successfully expressed in Sf9 insect cells by co-infection with baculoviruses respectively expressing the light and heavy chain of the antibodies. The purified antibodies are fully assembled as H2L2 (two heavy chains plus two light chains) heterodimer linked by covalent bonds. The b12 and VRC01 generated from insect cells reacted well to HIV-1 gp120, and their antigen binding ability is comparable to the mammalian cell-derived b12 as determined by ELISA and flow cytometry. Our data suggest that baculovirus/insect cell expression system can be utilized as an alternative to the mammalian expression system for the rapid production of HIV-1 broadly neutralizing antibodies.

A preliminary study of expression of human neonatal Fc-receptor (FcRn) in Escherichia coli

IntroductionHuman neonatal Fc-receptor is a potential novel antibody binding protein for development of immunoassay. Previous studies had shown that this human protein has high affinity to Ig G either in vivo or in vitro. However, none of the studies had attempted exploring the utility of human FcRn in antibody immobilisation strategy. In this study, we are in the direction towards development of a novel antibody binding protein by using human FcRn as an immobilisation platform. The three-dimensional structure of the protein was analysed. This protein was successful expressed in bacteria E. coli BL21 (DE3) by using expression vector pET-28b. The pET-28b vector contains poly-histidine tagged which allows detection and purification of expressed proteins. Objectives(1) To study the three dimensional structure of human FcRn, (2) To clone the human FcRn into pET-28b expression vector, (3) To express human FcRn in bacteria E. coli BL21 (DE3). MethodsHuman FcRn cDNA was ligated into pET-28b vector through EcoRI restriction site. The successful clone was transformed into E. coli BL21 (DE3) for expression. Expression in bacteria was induced by IPTG. Induction was conducted under 30°C and 37°C. Expression products were analysed by performing SDS-Page and Western Blot. The 3D structure of human FcRn was studied by using ViewerLite program. Results & DiscussionHuman FcRN was expressed under two sets of temperature, 30°C and 37°C, in 1 hour, 2 hours, 3 hours, and 6 hours. The induction for 3 hours showed highest amount of expressed products, under the above mentioned temperature. The amount of protein expressed in 6 hours of induction showed almost same thickness of band in SDS-PAGE compare to the induction under 3 hours period. ConclusionExpression of human FcRn can be conducted using bacteria expression system, instead of mammalian cell expression system which requires longer time and complicated process. Further study will be conducted to determine the antibody-binding activity and stability of the expressed protein.

The ZIP5 ectodomain co-localizes with PrP and may acquire a PrP-like fold that assembles into a dimer.

The cellular prion protein (PrPC) was recently observed to co-purify with members of the LIV-1 subfamily of ZIP zinc transporters (LZTs), precipitating the surprising discovery that the prion gene family descended from an ancestral LZT gene. Here, we compared the subcellular distribution and biophysical characteristics of LZTs and their PrP-like ectodomains. When expressed in neuroblastoma cells, the ZIP5 member of the LZT subfamily was observed to be largely directed to the same subcellular locations as PrPC and both proteins were seen to be endocytosed through vesicles decorated with the Rab5 marker protein. When recombinantly expressed, the PrP-like domain of ZIP5 could be obtained with yields and levels of purity sufficient for structural analyses but it tended to aggregate, thereby precluding attempts to study its structure. These obstacles were overcome by moving to a mammalian cell expression system. The subsequent biophysical characterization of a homogeneous preparation of the ZIP5 PrP-like ectodomain shows that this protein acquires a dimeric, largely globular fold with an α-helical content similar to that of mammalian PrPC. The use of a mammalian cell expression system also allowed for the expression and purification of stable preparations of Takifugu rubripes PrP-1, thereby overcoming a key hindrance to high-resolution work on a fish PrPC.

Expression of functionally active sialylated human erythropoietin in plants

Recombinant human erythropoietin (rhEPO), a glycohormone, is one of the leading biopharmaceutical products. The production of rhEPO is currently restricted to mammalian cell expression systems because of rhEPO's highly complex glycosylation pattern, which is a major determinant for drug-efficacy. Here we evaluate the ability of plants to produce different glycoforms of rhEPO. cDNA constructs were delivered to Nicotiana benthamiana (N. benthamiana) and transiently expressed by a viral based expression system. Expression levels up to 85 mg rhEPO/kg fresh leaf material were achieved. Moreover, co-expression of rhEPO with six mammalian genes required for in planta protein sialylation resulted in the synthesis of rhEPO decorated mainly with bisialylated N-glycans (NaNa), the most abundant glycoform of circulating hEPO in patients with anemia. A newly established peptide tag (ELDKWA) fused to hEPO was particularly well-suited for purification of the recombinant hormone based on immunoaffinity. Subsequent lectin chromatography allowed enrichment of exclusively sialylated rhEPO. All plant-derived glycoforms exhibited high biological activity as determined by a cell-based receptor-binding assay. The generation of rhEPO carrying largely homogeneous glycosylation profiles (GnGnXF, GnGn, and NaNa) will facilitate further investigation of functionalities with potential implications for medical applications.

A relatively low level of ribosome depurination by mutant forms of ricin toxin A chain can trigger protein synthesis inhibition, cell signaling and apoptosis in mammalian cells

The A chain of the plant toxin ricin (RTA) is an N-glycosidase that inhibits protein synthesis by removing a specific adenine from the 28S rRNA. RTA also induces ribotoxic stress, which activates stress-induced cell signaling cascades and apoptosis. However, the mechanistic relationship between depurination, protein synthesis inhibition and apoptosis remains an open question. We previously identified two RTA mutants that suggested partial independence of these processes in a yeast model. The goals of this study were to establish an endogenous RTA expression system in mammalian cells and utilize RTA mutants to examine the relationship between depurination, protein synthesis inhibition, cell signaling and apoptosis in mammalian cells. The non-transformed epithelial cell line MAC-T was transiently transfected with plasmid vectors encoding precursor (pre) or mature forms of wild-type (WT) RTA or mutants. PreRTA was glycosylated indicating that the native signal peptide targeted RTA to the ER in mammalian cells. Mature RTA was not glycosylated and thus served as a control to detect changes in catalytic activity. Both pre- and mature WT RTA induced ribosome depurination, protein synthesis inhibition, activation of cell signaling and apoptosis. Analysis of RTA mutants showed for the first time that depurination can be reduced by 40% in mammalian cells with minimal effects on inhibition of protein synthesis, activation of cell signaling and apoptosis. We further show that protein synthesis inhibition by RTA correlates more linearly with apoptosis than ribosome depurination.