KDEL Sequence Research Articles

Heat shock protein Gp96-Ig-peptide complexes based vaccine induce predominant immune responses at mucosal sites (46.13)

Abstract We have developed a vaccine design that uses the unique ability of the endoplasmic reticulum (ER) chaperone, heat shock protein gp96, to bind antigenic peptides and deliver them to antigen-presenting cells. Secreted form of gp96, gp96-Ig, was generated by replacing the ER retention sequence KDEL with the IgG1-Fc domain. To generate a cellular SIV vaccine, HEK-293 cells were transfected with gp96-Ig and the cDNAs encoding the SIV antigens gag, env and retanef. Irradiated, transfected 293 cells that secrete 1, 5 or 50 micrograms gp96-Ig-SIV complexes in 24h, were injected intraperitoneally (IP) in Mamu-A*01+ Rhesus Macaques at 0, 4 and 25 weeks. To determine whether gp96-Ig vaccination induces mucosal immunity, we used the 3T3-OVA-gp96-Ig/OT-1 model. Our data indicate that the best route of vaccination for induction of mucosal CD8-CTL immunity is the IP route. Gp96-Ig immunization increases frequency of CD11chighMHC classIIhighCD103+ cells in peritoneal cavity and efficiently induces CCR9 on responding T cells. In nonhuman primate model, the frequency of SIV-specific cells in the rectal mucosa reached 30-50% after the third immunization. We conclude that gp96-Ig vaccination startegy induces antigen-specific effector memory CD8T cells migrating to the intestinal mucosa and thus could be extremely useful for improving protection against a range of mucosal pathogens.

Phenotypic Knockout of CXCR4 by a Novel Recombinant Protein TAT/54R/KDEL Inhibits Tumors Metastasis

The chemokine receptor, CXCR4, and its specific ligand, CXCL12, have been proven to regulate the directional trafficking and invasion of breast cancer cells to sites of metastases, and similar phenomena have also been identified in many malignant tumors that aberrantly overexpress CXCR4. Therefore, blocking the interaction between CXCR4 and CXCL12 is considered a possible approach to efficiently prevent cancer metastasis. Employing a cellular phenotypic knockout strategy based on intrakines, we developed a novel recombinant chimeric protein, TAT/54R/KDEL, which contains three distinct functional domains: CXCL12/54R, a mutant of CXCL12 with CXCR4 antagonism, as well as HIV-derived TAT (47-57) and an endoplasmic reticulum retention four-peptide sequence KDEL that links at its NH(2) and COOH termini, respectively. Using the MOLT-4 cell line, which expressed CXCR4 highly and stably in vitro, we determined that TAT/54R/KDEL was able to efficiently transfer into the endoplasmic reticulum of tumor cells, where it specifically binds to the newly synthesized CXCR4 and prevents the latter from reaching the surface. Chemotaxis assays showed that the cells treated with TAT/54R/KDEL failed to migrate toward CXCL12. Furthermore, we observed that the systemic treatment of TAT/54R/KDEL could impair lung metastasis in a highly metastatic mammary cancer cell line, 4T1 cells, with the decrease of CXCR4 on their membrane. Our results suggest that the phenotypic knockout strategy of CXCR4 using a novel recombinant protein TAT/54R/KDEL might be a possible approach for inhibiting relative tumor metastasis mediated by CXCR4/CXCL12 interaction.

Calreticulin-dependent recycling in the early secretory pathway mediates optimal peptide loading of MHC class I molecules

Calreticulin is a lectin chaperone of the endoplasmic reticulum (ER). In calreticulin-deficient cells, major histocompatibility complex (MHC) class I molecules travel to the cell surface in association with a sub-optimal peptide load. Here, we show that calreticulin exits the ER to accumulate in the ER-Golgi intermediate compartment (ERGIC) and the cis-Golgi, together with sub-optimally loaded class I molecules. Calreticulin that lacks its C-terminal KDEL retrieval sequence assembles with the peptide-loading complex but neither retrieves sub-optimally loaded class I molecules from the cis-Golgi to the ER, nor supports optimal peptide loading. Our study, to the best of our knowledge, demonstrates for the first time a functional role of intracellular transport in the optimal loading of MHC class I molecules with antigenic peptide.

Auxin-binding proteins without KDEL sequence in the moss Funaria hygrometrica

Whereas the important plant growth regulator auxin has multiple effects in flowering plants, it induces a specific cell differentiation step in the filamentous moss protonema. Here, we analyse the presence of classical auxin-binding protein (ABP1) homologues in the moss Funaria hygrometrica. Microsomal membranes isolated from protonemata of F. hygrometrica have specific indole acetic acid-binding sites, estimated to be about 3–5 pmol/mg protein with an apparent dissociation constant (K d) between 3 and 5 μM. Western analyses with anti-ABP1 antiserum detected the canonical endoplasmic reticulum (ER)-localised 22–24 kDa ABP1 in Zea mays, but not in F. hygrometrica. Instead, polypeptides of 31–33 and 46 kDa were labelled in the moss as well as in maize. In F. hygrometrica these proteins were found exclusively in microsomal membrane fractions and were confirmed as ABPs by photo-affinity labelling with 5-azido-[7-3H]-indole-3-acetic acid. Unlike the classical corn ABP1, these moss ABPs did not contain the KDEL ER retention sequence. Consistently, the fully sequenced genome of the moss Physcomitrella patens, a close relative of F. hygrometrica, encodes an ABP1-homologue without KDEL sequence. Our study suggests the presence of putative ABPs in F. hygrometrica that share immunological epitopes with ABP1 and bind auxin but are different from the classical corn ABP1.

The Endoplasmic Reticulum Chaperone BiP/GRP78 Is Important in the Structure and Function of the Human Cytomegalovirus Assembly Compartment

We previously demonstrated that the endoplasmic reticulum (ER) chaperone BiP functions in human cytomegalovirus (HCMV) assembly and egress. Here, we show that BiP localizes in two cytoplasmic structures in infected cells. Antibodies to the extreme C terminus, which includes BiP's KDEL ER localization sequence, detect BiP in regions of condensed ER near the periphery of the cell. Antibodies to the full length, N terminus, or larger portion of the C terminus detect BiP in the assembly compartment. This inability of C-terminal antibodies to detect BiP in the assembly compartment suggests that BiP's KDEL sequence is occluded in the assembly compartment. Depletion of BiP causes the condensed ER and assembly compartments to dissociate, indicating that BiP is important for their integrity. BiP and pp28 are in association in the assembly compartment, since antibodies that detect BiP in the assembly compartment coimmunoprecipitate pp28 and vice versa. In addition, BiP and pp28 copurify with other assembly compartment components on sucrose gradients. BiP also coimmunoprecipitates TRS1. Previous data show that cells infected with a TRS1-deficient virus have cytoplasmic and assembly compartment defects like those seen when BiP is depleted. We show that a fraction of TRS1 purifies with the assembly compartment. These findings suggest that BiP and TRS1 share a function in assembly compartment maintenance. In summary, BiP is diverted from the ER to associate with pp28 and TRS1, contributing to the integrity and function of the assembly compartment.

Membrane topology of human NPC1L1, a key protein in enterohepatic cholesterol absorption

The Niemann-Pick C1 Like 1 (NPC1L1) is a predicted polytopic membrane protein that is critical for cholesterol absorption. NPC1L1 takes up free cholesterol into cells through vesicular endocytosis. Ezetimibe, a clinically used cholesterol absorption inhibitor, blocks the endocytosis of NPC1L1 thereby inhibiting cholesterol uptake. Human NPC1L1 is a 1,332-amino acid protein with a putative sterol-sensing domain (SSD) that shows sequence homo-logy to HMG-CoA reductase (HMGCR), Niemann-Pick C1 (NPC1), and SREBP cleavage-activating protein (SCAP). Here, we use protease protection and immunofluorescence in selectively permeabilized cells to study the topology of human NPC1L1. Our data indicate that NPC1L1 contains 13 transmembrane helices. The NH2-terminus of NPC1L1 is in the lumen while the COOH-terminus projects to the cytosol. human NPC1L1 contains seven small cytoplasmic loops--four small and three large luminal loops--one of which has been reported to bind ezetimibe. Ezetimibe-glucuronide, the major metabolite of ezetimibe in vivo, can block the internalization of NPC1L1 and cholesterol. The membrane topology of NPC1L1 is similar to that of NPC1, and the putative SSD of NPC1L1 is oriented in the same manner as those of HMGCR, NPC1, and SCAP. The defined topology of NPC1L1 provides necessary information for further dissecting the functions of the different domains of NPC1L1.

HPV16E7 tumor antigen modified by KDEL sequence induce specific cytotoxic T lymphocytes-dependent antitumor immunity

Infection by high-risk HPV (human papillomavirus) is the primary cause of cervical cancer. Dendritic cell-based (DC-based) therapeutic vaccine represents a promising approach to the prevention and treatment of many cancers, including HPV-related cancers, but current strategies have met with only limited success in preclinical and clinical research. It is necessary to find a properly and effective antigen presenting system of DC-based vaccine. To design a new HPV16 therapeutic vaccine using an endoplasmic reticulum (ER) retrieval signal and study its ability to induce the specific CTL activity in vitro and in vivo. E7(p)-KDEL and its control peptide were synthesized on solid phase. A series of methods were used, including standard (51)Cr-labeled release assay, enzyme-linked immunospot (ELISPOT) assay and ELISA, to detect the CTL activity induced by different peptides. Prophylactic models and therapeutic models were examined to detect the in vivo effectiveness of E7(p)-KDEL-loaded DCs. The specific CTL activity induced by E7(p)-KDEL-loaded DCs was much stronger than that induced by the other peptide-loaded DCs. Comparing with the control peptides, after incubation with the spleen cells of mice, the E7(p)-KDEL-loaded DCs could induce higher concentration of secreted IFN-gamma and had higher ELISPOT numbers. In animal models, E7(p)-KDEL-loaded DCs vaccines effectively protected mice against fatal TC-1 tumor challenge and cured tumor-bearing mice. The ER retrieval signal-mediated antigen delivery system may have important clinical application for cancer therapy, even virus infectious disease and autoimmune disease.

Ligand-independent Thrombopoietin Mutant Receptor Requires Cell Surface Localization for Endogenous Activity

The activating W515L mutation in the thrombopoietin receptor (MPL) has been identified in primary myelofibrosis and essential thrombocythemia. MPL belongs to a subset of the cytokine receptor superfamily that requires the JAK2 kinase for signaling. We examined whether the ligand-independent MPL(W515L) mutant could signal intracellularly. Addition of the endoplasmic reticulum (ER) retention KDEL sequence to the receptor C terminus efficiently locked MPL(W515L) within its natural ER/Golgi maturation pathway. In contrast to cells expressing the parental MPL(W515L), MPL(W515L)-KDEL-expressing FDC-P1 cells were unable to grow autonomously and to produce tumors in nude mice. When observed, tumor nodules resulted from in vivo selection of cells leaking the receptor at their surface. JAK2 co-immunoprecipitated with MPL(W515L)-KDEL but was not phosphorylated. We generated disulfide-bonded MPL(W515L) homodimers by the S402C substitution, both in the normal and KDEL context. Unlike MPL(W515L)-KDEL, MPL(W515L-S402C)-KDEL signaled constitutively and exhibited cell surface localization. These data establish that MPL(W515L) with appended JAK2 matures through the ER/Golgi system in an inactive conformation and suggest that the MPL(W515L)/JAK2 complex requires membrane localization for JAK2 phosphorylation, resulting in autonomous receptor signaling.

A novel recombinant protein TAT–GFP–KDEL with dual-function of penetrating cell membrane and locating at endoplasm reticulum

Although the potential value of phenotypic/functional knockout technology with intrabody/kine in prevention and cure of some serious diseases, such as AIDS and cancer, is being regarded, there are still several technical difficulties. One of the the most critical problems is how to directly deliver the intrabody/kine proteins into endoplasm reticulum (ER). In this study, a novel recombinant protein, TAT–GFP–KDEL, was designed and constructed. In this recombinant protein, HIV-derived TAT (47–57) and an ER retention four-peptide sequence KDEL were fused at the N-terminal and C-terminal of GFP respectively. The results showed that TAT–GFP–KDEL had been successfully expressed in bacteria BL21 and its purity reached to 95%. Moreover, we observed that this recombinant protein was able to efficiently transduce into MOLT-4 cells and accurately locate at ER. This study may provide an available strategy to promote the transmembrane delivery and ER localization of protein-based intrabody/kine.

Powerful mucosal immune response in macaques (Macaca mulatta) in response to SIV gp96-Ig immunization (39.35)

Abstract Induction of mucosal immunity is critical for protection from enteric pathogens. We have developed a vaccine design that utilizes the unique property of the endoplasmic reticulum chaperon, heat shock protein (HSP) gp96, to bind antigenic peptides and deliver them to APCs. The aim of our study was to evaluate safety and systemic and mucosal SIV-immunity with secreted gp96-Ig vaccines in non-human primates. We have generated a secreted form gp96 by replacing the ER retention sequence KDEL with the IgG1-Fc tag. HEK-293 cells were transfected with gp96-Ig and with the cDNAs encoding the SIV antigens gag, pol, env and retanef (reassorted rev, tat, nef). Irradiated, transfected 293 cells that secrete 1, 5 or 50 micrograms gp96-Ig-SIV-peptide complexes in 24h, were injected intraperitoneally in Mamu-A*01+ macaques at 0, 4 and 24 weeks. The frequency of SIVGag- and SIVTat- tetramer+ cells in the rectal mucosa reached 30-50% in some macaques after the third immunization. Tetramer+ cells expressed appropriate functional (granzymeB) and migration markers (alpha4beta7, CD103). The cells secrete IL-2 and IFN-gamma in response to different peptide (gag, tat, env) stimulation. We conclude that the cell secreted SIV-gp96-Ig vaccine is safe and can induce strong poly-specific, multifunctional CD8 responses in mucosal compartments that are thought to be critical for protection from SIV/HIV infection.

ERdj5 Sensitizes Neuroblastoma Cells to Endoplasmic Reticulum Stress-induced Apoptosis

Down-regulation of the unfolded protein response (UPR) can be therapeutically valuable in cancer treatment, and endoplasmic reticulum (ER)-resident chaperone proteins may thus be targets for developing novel chemotherapeutic strategies. ERdj5 is a novel ER chaperone that regulates the ER-associated degradation of misfolded proteins through its associations with EDEM and the ER stress sensor BiP. To investigate whether ERdj5 can regulate ER stress signaling pathways, we exposed neuroblastoma cells overexpressing ERdj5 to ER stress inducers. ERdj5 promoted apoptosis in tunicamycin, thapsigargin, and bortezomib-treated cells. To provide further evidence that ERdj5 induces ER stress-regulated apoptosis, we targeted Bcl-2 to ER of ERdj5-overexpressing cells. Targeting the Bcl-2 to ER prevented the apoptosis induced by ER stress inducers but not by non-ER stress apoptotic stimuli, suggesting induction of ER stress-regulated apoptosis by ERdj5. ERdj5 enhanced apoptosis by abolishing the ER stress-induced phosphorylation of eukaryotic translation initiation factor 2alpha (eIF2alpha) and the subsequent translational repression. ERdj5 was found to inhibit the eIF2alpha phosphorylation under ER stress through inactivating the pancreatic endoplasmic reticulum kinase. The compromised integrated stress response observed in ERdj5-overexpressing ER-stressed cells due to repressed eIF2alpha phosphorylation correlated with impaired neuroblastoma cell resistance under ER stress. These results demonstrate that ERdj5 decreases neuroblastoma cell survival by down-regulating the UPR, raising the possibility that this protein could be a target for anti-tumor approaches.

Change in glycosylation pattern with extension of endoplasmic reticulum retention signal sequence of mouse antibody produced by suspension-cultured tobacco BY2 cells

The production of antibodies using plants as bioreactors has been realized. Because sugar chain structures on recombinant proteins are a cause of concern, remodeling technology is highly promising. Localizing recombinant proteins in the endoplasmic reticulum (ER) affects the glycosylation pattern in transgenic plants. Previously, a mouse antibody produced by suspension-cultured tobacco BY2 cells has sugar chains with possible glycoepitopes as the predominant structures. In this study, we extended the Lys-Asp-Glu-Leu (KDEL) ER retention signal sequence over the heavy (H) and light (L) chains of the antibody and expressed the altered antibody in tobacco BY2 cells to study the effect of the KDEL sequence on glycosylation. For the antibody with the KDEL-extended H-chains, glycans with beta(1,2)-xylose or alpha(1,3)-fucose residues accounted for 49% of the total glycans. Meanwhile, for the antibody with the KDEL-extended H- and L-chains, glycans with xylose or fucose accounted for 38% of the total glycans. Although the addition of an ER retention signal shifted the dominant glycan structures of the KDEL-extended antibody to high-mannose-type structures, some of the antibodies escaped the retrieval system during intracellular traffic and were then modified by xylosylation or fucosylation.

Selection and characterization of KDEL-specific VHH antibody fragments and their application in the study of ER resident protein expression

Several diseases are caused by defects in the protein secretory pathway of the cell, particularly in the endoplasmic reticulum (ER). These defects are manifested by the activation of the unfolded protein response (UPR) that involves the transcriptional up-regulation of several ER resident proteins, the down-regulation of protein translation and up-regulation of ER associated degradation (ERAD). Although this transcriptional up-regulation of ER resident proteins during ER stress has been well described, data on differential protein expression of these same proteins are hardly available. Tools that would enable the simultaneous analysis of this set of proteins would be of high importance. Since the C-terminal KDEL sequence is a conserved epitope present in a large set of ER resident proteins, an antibody directed against this sequence would be such a tool. Using a carefully designed selection strategy, VHH antibody fragments from a non-immune phage display library were isolated that recognize the KDEL sequence at the C-terminus of proteins, irrespective of the protein context. In an accepted in vitro model for ER stress, this antibody was shown to be an excellent tool to study differences in ER resident protein expression. Furthermore, the application of this antibody showed differences in ER resident protein levels during replicative senescence of human umbilical vein endothelial cells (HUVECs), underlining its significance in biological research. The selection strategy used to obtain these KDEL-specific antibodies opens up ways to select antibodies to other conserved epitopes, such as the nuclear localization signal (NLS) or the peroxisomal targeting sequence, permitting the simultaneous analysis of specific groups of proteins.

Functional knockdown of VCAM-1 at the posttranslational level with ER retained antibodies

Vascular cell adhesion molecule 1 (VCAM-1) is involved in the recruitment of leukocytes to inflammatory sites. In this study we present the first functional knockdown of VCAM-1 using an ER retained antibody construct. We generated a knockdown construct encoding the VCAM-1 specific single chain variable fragment scFv6C7.1 fused to the C-terminal ER retention sequence KDEL. HEK-293:VCAM-YFP cells stably expressing a VCAM-YFP fusion protein were transiently transfected with the knockdown construct and showed down-regulation of surface VCAM-1. Knockdown efficiency of the system is time-dependent due to used transient transfection of the intrabody construct. Furthermore, intrabody mediated knockdown of HEK-293:VCAM-YFP cells also impaired cell–cell interaction with Jurkat cells that are endogenously expressing VLA-4, the physiological partner of VCAM-1. Posttranslational knockdown with ER retained antibodies seems to be a promising technique, as shown here for VCAM-1.

Ligand-Independent MPLW515L Activity Requires Cell Surface Localization

The activating mutation W515L in the thrombopoietin receptor Mpl has been identified in a minority of primitive myelofibrosis and essential thrombocythemia negative for the JAK2V617F mutation. Mpl and the erythropoietin receptor EpoR belong to a distinct subset of the cytokine receptor superfamily. Mpl and EpoR lack intrinsic kinase activity and rely on the activation of the cytoplasmic janus tyrosine kinases (JAKs) for signal transduction. Recently, it was shown that EpoR dimerizes in a ligand-independent fashion in the endoplasmic reticulum (ER), where it associates with JAK2. Normal receptor signaling occurs at the cell surface after ligand binding, believed to trigger changes in the dimeric receptor conformation allowing for JAK2 trans-phosphorylation and activation. Based on numerous functional and structural similarities between Mpl and EpoR, we examined the possible occurrence of abnormal intracellular signaling concerning the endogenous ligand-independent MplW515L that might be involved in myeloproliferative disorder pathogenesis. The murine factor-dependent FDC-P1 cell line was transduced with human HA-tagged MplW515L modified with the ER retention KDEL sequence, in order to prevent its cell surface expression, or with the human HA-tagged MplW515L, as a positive control. We isolated cells expressing either the normally-processed Mpl mutant or MplW515L locked within its natural ER/Golgi maturation pathway. MplW515L-KDEL was unable to support autonomous growth of the cytokine-dependent FDC-P1 cells. Co-immunoprecipitation analysis showed that MplW515L-KDEL associated with JAK2 in the ER without activating the kinase. Indeed, MplW515L-KDEL-expressing cells lacked both constitutive and TPO-dependent phosphorylation of STAT5, ERK1/2 and AKT signaling molecules. Moreover, FDC-P1 cells expressing the ER-retained MplW515L became tumorigenic in nude mice only after in vivo selection of cells leaking the receptor at their surface. Finally, covalent disulfide modification (S402C) of MplW515L-KDEL, resulting in homodimerization of the receptor led to autonomous FDC-P1 cell growth. MplW515L-S402C-KDEL exhibited enhanced cell surface localization, suggesting that homodimerization overrided the ER/Golgi retention induced by the KDEL motif. Our results indicated that forced dimerization might promote cell surface localization. We propose that MplW515L is bound to JAK2 intracellularly in the ER/Golgi in an inactive conformation. In order to induce ligand-independent phosphorylation and activation of JAK2 and consequently downstream signaling, the MplW515L mutant receptor strictly needs to reach the cell surface to probably acquire a dimeric conformation.

Expression of anti-K99 scFv in transgenic rice tissues and its functional characterization

As a first step towards manufacturing functional anti-K99 single chain variable antibody fragment (scFv) in a plant system to prevent colibacillosis in neonatal calves, we investigated the feasibility of producing these antibodies in rice plants. Two scFv constructs, with or without the endoplasmic reticulum (ER) targeting KDEL sequence, were introduced into rice for either ER-retention of the recombinant antibody or its secretion. In agreement with several other published reports, extremely low-levels of scFv were produced in rice plants transformed with the construct lacking the ER-targeting sequence. Constructs containing the KDEL sequence resulted in significantly higher levels of the antibody in rice leaves. Although scFv transcripts were found in all three rice tissues analyzed, scFv protein was detected only in the leaf and embryo tissues and not in the endosperm portion of the seed. Functionality of the rice-produced scFv was tested in two in vitro assays, i.e., inhibition of K99-induced horse red blood cell agglutination and inhibition of the attachment of enterotoxigenic Escherichia coli (ETEC) to calf enterocytes. Rice-scFv was found to be functionally equivalent to anti-K99 monoclonal antibody (mAb) in both the assays. The results obtained in this investigation provide valuable information and in combination with other studies of this kind, will be helpful in devising strategies to improve production of useful recombinant proteins in the seeds.

D1 and D2 Dopamine Receptor Expression Is Regulated by Direct Interaction with the Chaperone Protein Calnexin

As for all proteins, G protein-coupled receptors (GPCRs) undergo synthesis and maturation within the endoplasmic reticulum (ER). The mechanisms involved in the biogenesis and trafficking of GPCRs from the ER to the cell surface are poorly understood, but they may involve interactions with other proteins. We have now identified the ER chaperone protein calnexin as an interacting protein for both D(1) and D(2) dopamine receptors. These protein-protein interactions were confirmed using Western blot analysis and co-immunoprecipitation experiments. To determine the influence of calnexin on receptor expression, we conducted assays in HEK293T cells using a variety of calnexin-modifying conditions. Inhibition of glycosylation either through receptor mutations or treatments with glycosylation inhibitors partially blocks the interactions with calnexin with a resulting decrease in cell surface receptor expression. Confocal fluorescence microscopy reveals the accumulation of D(1)-green fluorescent protein and D(2)-yellow fluorescent protein receptors within internal stores following treatment with calnexin inhibitors. Overexpression of calnexin also results in a marked decrease in both D(1) and D(2) receptor expression. This is likely because of an increase in ER retention because confocal microscopy revealed intracellular clustering of dopamine receptors that were co-localized with an ER marker protein. Additionally, we show that calnexin interacts with the receptors via two distinct mechanisms, glycan-dependent and glycan-independent, which may underlie the multiple effects (ER retention and surface trafficking) of calnexin on receptor expression. Our data suggest that optimal receptor-calnexin interactions critically regulate D(1) and D(2) receptor trafficking and expression at the cell surface, a mechanism likely to be of importance for many GPCRs.

Aberrant localization and underglycosylation of highly accumulating single-chain Fv-Fc antibodies in transgenic Arabidopsis seeds.

Production of high-value recombinant proteins in transgenic seeds is an attractive and economically feasible alternative to conventional systems based on mammalian cells and bacteria. In contrast to leaves, seeds allow high-level accumulation of recombinant proteins in a relatively small volume and a stable environment. We demonstrate that single-chain variable fragment (scFv)-Fc antibodies, with N-terminal signal sequence and C-terminal KDEL tag, can accumulate to very high levels as bivalent IgG-like antibodies in Arabidopsis thaliana seeds and illustrate that a plant-produced anti-hepatitis A virus scFv-Fc has similar antigen-binding and in vitro neutralizing activities as the corresponding full-length IgG. As expected, most scFv-Fc produced in seeds contained only oligomannose-type N-glycans, but, unexpectedly, 35-40% was never glycosylated. A portion of the scFv-Fc was found in endoplasmic reticulum (ER)-derived compartments delimited by ribosome-associated membranes. Additionally, consistent with the glycosylation data, large amounts of the recombinant protein were deposited in the periplasmic space, implying a direct transport from the ER to the periplasmic space between the plasma membrane and the cell wall. Aberrant localization of the ER chaperones calreticulin and binding protein (BiP) and the endogenous seed storage protein cruciferin in the periplasmic space suggests that overproduction of recombinant scFv-Fc disturbs normal ER retention and protein-sorting mechanisms in the secretory pathway.

Extended‐release of Flt intraceptors in the cornea inhibit injury‐induced corneal angiogenesis

We constructed non-toxic albumin nanoparticles with a plasmid coding for Flt intraceptor comprising domains 2–3 linked to the endoplasmic retention sequence KDEL (pCMV.Flt23K). The nanoparticles entered into the Balb/C mice corneal cytoplasm as determined by confocal microscopy and transmission electron microscopy. FITC-conjugated nanoparticles were injected into the cornea, and fluorescence was observed for 4 weeks. Flt23K was expressed by naked plasmids in corneas for 8 days and by the nanoparticles for 5 weeks. Mouse corneas injected with pCMV.Flt23K-loaded nanoparticles 3 weeks prior to mechanical-alkali trauma developed a mean percentage of corneal neovascularization of 35.0±6.0% as compared to naked pCMV.Flt23K (58.3±8.7%), empty pCMV nanoparticles (55.4 ± 2.7%), or saline (61.6±10.5%) (p’s <0.05) (n = 7) two weeks after injury. Experiments are being conducted for permanent, stable production of Flt23K (and also soluble Flt-1) with the non-viral integrating vector, piggyBac, to allow for longer and more pronounced suppression of corneal neovascularization.

Abrogation of Viral Interleukin-6 (vIL-6)-Induced Signaling by Intracellular Retention and Neutralization of vIL-6 with an Anti-vIL-6 Single-Chain Antibody Selected by Phage Display

Human herpesvirus 8 (HHV-8) encodes several putative oncogenes, which are homologues to cellular host genes known to function in cell cycle regulation, control of apoptosis, and cytokine signaling. Viral interleukin (vIL-6) is believed to play an important role in the pathogenesis of Kaposi's sarcoma as well as primary effusion lymphoma and multicentric Castleman's disease. Therefore, vIL-6 is a promising target for novel therapies directed against HHV-8-associated diseases. By phage display screening of human synthetic antibody libraries, we have selected a specific recombinant antibody, called monoclonal anti-vIL-6 (MAV), binding to vIL-6. The epitope recognized by MAV was localized on the top of the D helix of the vIL-6 protein, which is a part of receptor binding site III. Consequently, MAV specifically inhibits vIL-6-mediated growth of the primary effusion lymphoma-derived cell line BCBL-1 and blocks STAT3 phosphorylation in the human hepatoma cell line HepG2. Since it was previously found that vIL-6 can also induce signals from within the cell, presumably within the endoplasmic reticulum, we fused the recombinant antibody MAV with the endoplasmic retention sequence KDEL (MAV-KDEL). As a result, COS-7 cells expressing MAV-KDEL and synthesizing vIL-6 ceased to secrete the cytokine. Moreover, we observed that vIL-6 that was bound to MAV-KDEL and retained in the endoplasmic reticulum did not induce STAT3 phosphorylation in HepG2 cells. We conclude that the activity of the intracellularly retained vIL-6 protein is neutralized by MAV-KDEL. Our results might represent a novel therapeutic strategy to neutralize virally encoded growth factors or oncogenes.