A photoaffinity labeling strategy identified EF1A1 as a binding protein of cyclic dinucleotide 2'3'-cGAMP.

2'3'-cyclic GMP-AMP (2'3'-cGAMP), generated by cyclic GMP-AMP synthase (cGAS) under activation by cytosolic DNA, has a vital role in innate immune response via its receptor protein stimulator of interferon genes (STING) to fight viral infections and tumors. In order to have a complete understanding of biological functions of 2'3'-cGAMP, it is important to find out whether 2'3'-cGAMP has other unrevealed binding proteins present in mammalian cells and executes unknown functions. Here we report the 2'3'-cGAMP-based photoaffinity probes that capture and isolate 2'3'-cGAMP-binding proteins. These probes enable the identification of some potential 2'3'-cGAMP-binding proteins from HeLa cells. EF1A1, an essential protein regulating protein synthesis, is further validated to associate with 2'3'-cGAMP in vitro and in cells to impede protein synthesis. Thus, our studies provide a powerful approach to enable identification of the 2'3'-cGAMP interactome, discover unknown functions of 2'3'-cGAMP, and understand its physiological/pathological roles in tumor immunity and immune-related diseases.

Recombinant elongation factor 1 alpha of Haemonchus contortus affects the functions of goat PBMCs.

Excretory/secretory proteins of Haemonchus contortus (HcESPs) intermingle comprehensively with host immune cells and modulate host immune responses. In this study, H contortus ES antigen named as elongation factor 1 alpha (HcEF-1α) was cloned and expressed. The influences of recombinant HcEF-1α on multiple functions of goat peripheral blood mononuclear cells (PBMCs) were observed in vitro. Immunoblot analysis revealed that rHcEF-1α was recognized by the serum of goat infected with H contortus. Immunofluorescence analysis indicated that rHcEF-1α was bound on surface of PBMCs. Moreover, the productions of IL-4, TGF-ß1, IFN-γ and IL-17 of cells were significantly modulated by the incubation with rHcEF-1α. The production of interleukin IL-10 was decreased. Cell migration, cell proliferation and cell apoptosis were significantly increased; however, nitric oxide production (NO) was significantly decreased. The MHC II molecule expression of cells incubated with rHcEF-1α was increased significantly, whereas MHC-I was not changed as compared to the control groups (PBS control and pET32a). These findings indicated that rHcEF-1α protein might play essential roles in functional regulations of HcESPs on goat PBMC and mediate the immune responses of the host during host-parasite relationship.

Liposomal Elongation Factor-1α Triggers Effector CD4 and CD8 T Cells for Induction of Long-Lasting Protective Immunity against Visceral Leishmaniasis.

Despite advances, identification and formulation of safe and effective vaccine for long-lasting protection against leishmaniasis is still inadequate. In this study, we have identified a novel antigen, leishmanial elongation factor-1α (EF1-α), as an immunodominant component of solubilized leishmanial membrane antigens that reacts with visceral leishmaniasis (VL) sera and induces cellular proliferative and cytokine response in PBMCs of cured VL subjects. Leishmanial EF1-α is a 50 kDa antigen that plays a crucial role in pathogen survival by regulating oxidative burst in the host phagocytes. Previously, immunodominant truncated forms of EF1-α from different species of Leishmania have been reported. Formulation of the L. donovani 36 kDa truncated as well as the cloned recombinant EF1-α in cationic liposomes induce strong resistance to parasitic burden in liver and spleen of BALB/c mice through induction of DTH and a IL-10 and TGF-ß suppressed mixed Th1/Th2 cytokine responses. Multiparametric analysis of splenocytes for generation of antigen-specific IFN-γ, IL2, and TNF-α producing lymphocytes indicate that cationic liposome facilitates expansion of both CD4+ as well as CD8+ memory and effector T cells. Liposomal EF1-α is a novel and potent vaccine formulation against VL that imparts long-term protective responses. Moreover, the flexibility of this formulation opens up the scope to combine additional adjuvants and epitope selected antigens for use in other disease forms also.

The Eukaryotic Elongation Factor 1 Alpha (eEF1α) from the Parasite Leishmania infantum Is Modified with the Immunomodulatory Substituent Phosphorylcholine (PC).

Proteins and glycolipids have been found to be decorated with phosphorylcholine (PC) both in protozoa and nematodes that parasitize humans and animals. PC epitopes can provoke various effects on immune cells leading to an immunomodulation of the host's immune system that allows long-term persistence of the parasites. So far, only a limited number of PC-modified proteins, mainly from nematodes, have been identified. Infections caused by Leishmania spp. (e.g., L. infantum in southern Europe) affect about 12 million people worldwide and are characterized by a wide spectrum of clinical forms in humans, ranging from cutaneous to fatal visceral leishmaniasis. To establish and maintain the infection, these protozoa are dependent on the secretion of effector molecules into the host for modulating their immune system. In this project, we analyzed the PC modification of L. infantum promastigotes by 2D-gel based proteomics. Western blot analysis with the PC-specific antibody TEPC-15 revealed one PC-substituted protein in this organism, identified as eEF1α. We could demonstrate that the binding of eEF1α to one of its downstream effectors is dependent on its PC-modification. In this study we provide evidence that in this parasite the modification of eEF1α with PC may be essential for its function as an important virulence factor.

Vaccination with Eimeria tenella elongation factor-1α recombinant protein induces protective immunity against E. tenella and E. maxima infections.

Avian coccidiosis is caused by multiple species of the apicomplexan protozoan, Eimeria, and is one of the most economically devastating enteric diseases for the poultry industry worldwide. Host immunity to Eimeria infection, however, is relatively species-specific. The ability to immunize chickens against different species of Eimeria using a single vaccine will have a major beneficial impact on commercial poultry production. In this paper, we describe the molecular cloning, purification, and vaccination efficacy of a novel Eimeria vaccine candidate, elongation factor-1α (EF-1α). One day-old broiler chickens were given two subcutaneous immunizations one week apart with E. coli-expressed E. tenella recombinant (r)EF-1α protein and evaluated for protection against challenge infection with E. tenella or E. maxima. rEF-1α-vaccinated chickens exhibited increased body weight gains, decreased fecal oocyst output, and greater serum anti-EF-1α antibody levels following challenge infection with either E. tenella or E. maxima compared with unimmunized controls. Vaccination with EF-1α may represent a new approach to inducing cross-protective immunity against avian coccidiosis in the field.

Immunoproteomic Identification of p29 Antigen as the Elongation Factor-1α of Leishmania mexicana.

Previously, we identified five Leishmania mexicana antigens reacting with antibodies from cutaneous leishmaniasis patients, designated on the basis of their molecular weights as p26 (pI 7.8), p27 (pI 8.1), p28 (pI 8.6), p29 (pI 8.5), and p31 (pI 9.0). Among these antigens, p29 was most strongly recognized by the antibodies. Thereafter, p29 was identified as elongation factor-1α (EF-1α) of Leishmania mexicana through mass spectrometry analysis and western blot using a commercial antibody that reacted with EF-1α from different species. Our results showed that the p29 antigen of Leishmania mexicana is EF-1α.

Characterization and identification of calmodulin and calmodulin binding proteins in hemocyte of the black tiger shrimp (Penaeus monodon).

Calmodulin (CaM), a ubiquitous intracellular calcium (Ca(2+)) sensor in all eukaryotic cells, is one of the well-known signaling proteins. Previously, CaM gene has shown a high transcriptional level in hemocyte of the pathogen infected shrimp, suggesting that shrimp CaM does not only regulate Ca(2+) metabolism, but is also involved in immune response cascade. In the present study, the CaM gene of shrimp Penaeus monodon was identified and the recombinant P.monodon CaM (rPmCaM) was produced and biochemically characterized. The identification of CaM-binding proteins was also performed. The PmCaM cDNA consisted of an open reading frame of 447 bp encoding for 149 amino acid residues with a calculated mass of 16,810 Da and an isoelectric point of 4.09. Tissue distribution showed that the PmCaM transcript was expressed in all examined tissues. The results of gel mobility shift assay, circular dichroism spectroscopy and fluorescence spectroscopy all confirmed that the conformational changes of the rPmCaM were observed after the calcium binding. According to the gene silencing of PmCaM transcript levels, the shrimp's susceptibility to pathogenic Vibrio harveyi infection increased in comparison with that of the control groups. Protein pull-down assay and LC-MS/MS analysis were performed to identify rPmCaM-binding proteins involved in shrimp immune responses and transglutaminase, elongation factor 1-alpha, elongation factor 2 and actin were found. However, by computational analysis, only the first three proteins contained CaM-binding domain. These findings suggested that PmCaM may play an important role in regulation of shrimp immune system.

Identification of novel autoantibodies in type 1 diabetic patients using a high-density protein microarray.

Autoantibodies can facilitate diagnostic and therapeutic means for type 1 diabetes (T1DM). We profiled autoantibodies from serum samples of 16 T1DM patients, 16 type 2 diabetic (T2DM) patients, and 27 healthy control subjects with normal glucose tolerance (NGT) by using protein microarrays containing 9,480 proteins. Two novel autoantibodies, anti-EEF1A1 and anti-UBE2L3, were selected from microarrays followed by immunofluorescence staining of pancreas. We then tested the validity of the candidates by ELISA in two independent test cohorts: 1) 95 adults with T1DM, 49 with T2DM, 11 with latent autoimmune diabetes in adults (LADA), 20 with Graves disease, and 66 with NGT and 2) 33 children with T1DM and 34 healthy children. Concentrations of these autoantibodies were significantly higher in T1DM patients than in NGT and T2DM subjects (P < 0.01), which was also confirmed in the test cohort of children (P < 0.05). Prevalence of anti-EEF1A1 and anti-UBE2L3 antibodies was 29.5% and 35.8% in T1DM, respectively. Of note, 40.9% of T1DM patients who lack anti-GAD antibodies (GADA) had anti-EEF1A1 and/or anti-UBE2L3 antibodies. These were also detected in patients with fulminant T1DM but not LADA. Our approach identified autoantibodies that can provide a new dimension of information indicative of T1DM independent of GADA and new insights into diagnosis and classification of T1DM.

[Preparation of monospecific antibodies against isoform 2 of translation elongation factor 1A (eEF1A2)].

Amino acid sequences of eukaryotic translation elongation factor isoform 1 (eEF1A1) and 2 (eEF1A2) were compared and two peptide fragments of eEF1A2 were chosen as linear antigenic determinants for generation of monospecific antipeptide antibodies. Selected peptides were synthesized, conjugated to bovine serum albumin (BSA) and used for mice immunizations. Antibodies, produced against the eEF1A2 fragment 330-343 conjugated to BSA, specifically recognized this isoform in the native and partially denatured states but did not interact with the eEF1A1 isoform. It was shown that these monospecific anti-eEF1A2 antibodies could be employed for eEF1A2 detection both by enzyme-linked immunosorbent assay and by immunoblotting.

Elongation factor-1α is a novel protein associated with host cell invasion and a potential protective antigen of Cryptosporidium parvum.

The phylum Apicomplexa comprises obligate intracellular parasites that infect vertebrates. All invasive forms of Apicomplexa possess an apical complex, a unique assembly of organelles localized to the anterior end of the cell and involved in host cell invasion. Previously, we generated a chicken monoclonal antibody (mAb), 6D-12-G10, with specificity for an antigen located in the apical cytoskeleton of Eimeria acervulina sporozoites. This antigen was highly conserved among Apicomplexan parasites, including other Eimeria spp., Toxoplasma, Neospora, and Cryptosporidium. In the present study, we identified the apical cytoskeletal antigen of Cryptosporidium parvum (C. parvum) and further characterized this antigen in C. parvum to assess its potential as a target molecule against cryptosporidiosis. Indirect immunofluorescence demonstrated that the reactivity of 6D-12-G10 with C. parvum sporozoites was similar to those of anti-ß- and anti-γ-tubulins antibodies. Immunoelectron microscopy with the 6D-12-G10 mAb detected the antigen both on the sporozoite surface and underneath the inner membrane at the apical region of zoites. The 6D-12-G10 mAb significantly inhibited in vitro host cell invasion by C. parvum. MALDI-TOF/MS and LC-MS/MS analysis of tryptic peptides revealed that the mAb 6D-12-G10 target antigen was elongation factor-1α (EF-1α). These results indicate that C. parvum EF-1α plays an essential role in mediating host cell entry by the parasite and, as such, could be a candidate vaccine antigen against cryptosporidiosis.

Elongation factor 1-alpha is released into the culture medium during growth of Giardia intestinalis trophozoites.

The molecular pathogenesis of the intestinal parasite Giardia intestinalis is still not fully understood but excretory-secretory products have been suggested to be important during host-parasite interactions. Here we used SDS-PAGE gels and MALDI-TOF analysis to identify proteins released by Giardia trophozoites during in vitro growth. Serum proteins (mainly bovine serum albumin) in the growth medium, bind to the parasite surface and they are continuously released, which interfere with parasite secretome characterization. However, we identified two released Giardia proteins: elongation factor-1 alpha (EF-1α) and a 58 kDa protein, identified as arginine deiminase (ADI). This is the first description of EF-1α as a released/secreted Giardia protein, whereas ADI has been identified in an earlier secretome study. Two genes encoding EF-1α were detected in the Giardia WB genome 35 kbp apart with almost identical coding sequences but with different promoter and 3' regions. Promoter luciferase-fusions showed that both genes are transcribed in trophozoites. The EF-1α protein localizes to the nuclear region in trophozoites but it relocalizes to the cytoplasm during host-cell interaction. Recombinant EF-1α is recognized by serum from giardiasis patients. Our results suggest that released EF-1α protein can be important during Giardia infections.

The prognostic significance of elongation factor eEF1A2 in ovarian cancer.

OBJECTIVE: To determine whether eukaryotic elongation factor 1 alpha 2 (eEF1A2), a transforming gene previously shown to be highly expressed in primary human ovarian tumours, is a prognostic marker. METHODS: We have used an antibody specific for eEF1A2 to measure eEF1A2 protein expression in 500 primary ovarian tumours in a tissue microarray. We have also ectopically expressed eEF1A2 in SK-OV-3 cells, a clear cell carcinoma line that does not normally express eEF1A2. RESULTS: We have shown that eEF1A2 has high expression levels in approximately 30% of all primary ovarian tumours. 50% of serous tumours, 30% of endometrioid, 19% of mucinous and 8% of clear cell tumours highly express eEF1A2. Ectopic expression of eEF1A2 in the SK-OV-3 clear cell carcinoma line enhances their in vitro proliferative capacity and ability to form tumour-like spheroids in hanging drop culture. Expression of eEF1A2 did not alter sensitivity to anoikis, cisplatin, or taxol. In serous cancer, eEF1A2 is an independent prognostic marker for survival and high eEF1A2 protein expression was associated with increased probability of 20-year survival. CONCLUSIONS: eEF1A2 is highly expressed in ovarian carcinomas. Its expression enhances cell growth in vitro, and eEF1A2 expression is likely to be a useful ovarian cancer prognostic factor in ovarian cancer patients with serous tumours.

Effect of bacterial endotoxin LPS on expression of INF-gamma and IL-5 in T-lymphocytes from asthmatics.

Epidemiological evidence, in vitro studies and animal models suggest that exposure to the bacterial endotoxin lipopolysaccharide (LPS) can influence the development and severity of asthma. Although it is known that signaling through Toll-like receptors (TLR) is required for adaptive T helper cell type 1 and 2 responses, it is unclear whether the LPS ligand TLR 4 is expressed on CD4(+) and CD8(+) T-lymphocytes and if so, whether LPS could modulate the T(H)1 or T(H)2 response in this context. The present authors have, therefore, examined the expression of TLR 4 on peripheral blood CD4(+) and CD8(+) T-lymphocytes using RT-PCR method and FACS analyses. Furthermore, the authors have studied the IL-12-induced expression of the T(H)1-associated cytokine INF-gamma and the IL-4-induced expression of the T(H)2-specific cytokine IL-5 in the presence of LPS using ELISA and compared nine atopic asthmatic subjects and eleven nonatopic normal volunteers. There was an increased anti-CD3/anti-CD28-induced IL-5 expression in T cells of asthmatics compared with normals (p<0.01). In the presence of IL-4 (10 ng/ml), there was an additional increase in IL-5 expression and this additional increase was greater in T cells of normals compared with asthmatics (p<0.05). There was an expression of INF-gamma in anti-CD3/anti-CD28-induced T-lymphocytes without differences between both groups (NS). In the presence of IL-12 (10 ng/ml), there was an increase in INF-gamma release without differences between normals and asthmatics (NS). In the presence of different concentrations of LPS (10 ng/ml, 1 mug/ml), there was a decrease in IL-4-induced IL-5 expression without differences in both groups, indicating an intact T(H)2 response to bacterial endotoxin LPS in asthma. Interestingly, LPS increased the IL-12-induced INF-gamma release in a concentration-dependent manner in T-lymphocytes of normals but this could not be found in T cells of asthmatics, indicating an impaired T(H)1 response to bacterial endotoxin LPS in asthma. In addition, there was a TLR 4 expression on CD4(+) T-lymphocytes of normals and to a lesser extent in asthmatics but this TLR 4 expression could not be found on CD8(+) T cells of both groups. In conclusion, there may be an impaired concentration-dependent LPS-induced T(H)1 rather than a T(H)2 response in allergic adult asthmatics compared with normal volunteers. One reason for this could be a reduced TLR 4 expression on CD4(+) T-lymphocytes of asthmatic subjects.

Differential rejection of salmon lice by pink and chum salmon: disease consequences and expression of proinflammatory genes.

The consequences of high (735 copepodids fish-1) and low (243 copepodids fish-1) level exposures of size-matched juvenile pink and chum salmon to Lepeophtheirus salmonis copepodids were examined. At both levels of exposure the prevalence and abundance of L. salmonis was significantly higher on chum salmon. In addition, the weight of exposed chum salmon following the high exposure was significantly less than that of unexposed chum salmon. At both exposures, the haematocrit of exposed chum salmon was significantly less than that of unexposed chum. Neither weight nor haematocrit of pink salmon was affected by exposures at these levels. Despite the presence of microscopic inflammatory lesions associated with attachment of L. salmonis on the epithelium of gill and fin of both salmon species, there were no mortalities following either exposure. A transient cortisol response was observed in chum salmon 21 d after low exposure. An earlier and quantitatively higher expression of the proinflammatory genes interleukin-8 (IL-8), tumour necrosis factor alpha-1 (TNFalpha-1) and interleukin-1beta (IL-1beta) in fin and head kidney of pink salmon suggested a mechanism of more rapid louse rejection in this species. Together, these observations indicate a relatively enhanced innate resistance to L. salmonis in the juvenile pink salmon compared with the juvenile chum salmon.

Translation elongation factor eEF1A2 is essential for post-weaning survival in mice.

Translation elongation factor eEF1A, formerly known as EF-1 alpha, exists as two variant forms; eEF1A1, which is almost ubiquitously expressed, and eEF1A2, whose expression is restricted to muscle and brain at the level of whole tissues. Expression analysis of these genes has been complicated by a general lack of availability of antibodies that specifically recognize each variant form. Wasted mice (wst/wst) have a 15.8-kilobase deletion that abolishes activity of eEF1A2, but before this study it was unknown whether the deletion also affected neighboring genes. We have generated a panel of anti-peptide antibodies and used them to show that eEF1A2 is expressed at high levels in specific cell types in tissues previously thought not to express this variant, such as pancreatic islet cells and enteroendocrine cells in colon crypts. Expression of eEF1A1 and eEF1A2 is shown to be generally mutually exclusive, and we relate the expression pattern of eEF1A2 to the phenotype seen in wasted mice. We then carried out a series of transgenic experiments to establish whether the expression of other genes is affected by the deletion in wasted mice. We show that aspects of the phenotype such as motor neuron degeneration relate precisely to the relative expression of eEF1A1 and eEF1A2, whereas the immune system abnormalities are likely to result from a stress response. We conclude that loss of eEF1A2 function is solely responsible for the abnormalities seen in these mice.

cDNA cloning and immunologic characterization of a novel EF-1beta allergen from Penicillium citrinum.

BACKGROUND: We have identified previously that Penicillium citrinum is the most prevalent Penicillium species in the Taipei area. It is important to delineate the whole spectrum of allergenic components of this prevalent airborne fungus. The purpose of this study was to identify novel P. citrinum allergens through molecular cloning of allergen genes using a cDNA library of P. citrinum and sera from patients with bronchial asthma. METHODS: A lambda-Uni-ZAP XR-based cDNA library of P. citrinum was screened with sera from asthmatic patients. An IgE-binding cDNA clone was isolated and heterologously expressed in Escherichia coli. The frequency of IgE-binding to the expressed protein and the IgE reactivity to allergen subunits were analyzed by immunoblotting. RESULTS: An IgE-reactive cDNA clone (clone B) was isolated by plaque immunoassay. The cDNA insert is 876-bp long and encodes a 228-amino acid polypeptide with a calculated molecular mass of 25 035 Da. Protein database search with the deduced clone B sequence revealed that 121 (53%) and 82 (36%) of the 228 amino acids were identical to those of the elongation factor 1-beta (EF-1beta) proteins from the yeast Saccharomyces cerevisiae and the parasite Echinococcus granulosus, respectively. His-tagged recombinant clone B proteins were constructed and expressed in E. coli. Seven (8%) of the 92 serum samples from patients with bronchial asthma showed IgE-binding to the recombinant clone B protein. Among these seven positive sera, five demonstrated IgE-binding to the C-terminal fragment (aa 119-228) while the other two sera showed IgE reactivity to the N-terminal fragment (aa 1-118) of this newly identified EF-1betaPenicillium allergen. CONCLUSIONS: A novel P. citrinum allergen (Pen c 24) was identified and characterized in the present study. Results obtained provide more information about allergens of prevalent airborne fungi and a basis to understand more about the IgE responses in human atopic disorders and in parasitic infections.

Cloning of a human antibody directed against human neuroblastoma cells and specific for human translation elongation factor 1alpha.

Human sera have shown antitumor effects mediated by tumor-specific immunoglobulin M (IgM) antibodies. Most people who have cytotoxic serum are in good health and show no evidence of exposure to tumor antigens. We characterized the serum of a healthy female adult that was highly lytic to a neuroblastoma cell line via IgM-activated complement (>60% of malignant cells were killed during the 60-min assay). Complement-dependent lysis was not mediated by other classes of serum antibodies (data not shown) which is consistent with the findings of Ollert et al. To identify the target antigen on neuroblastoma cells, we fractionated neuroblastoma cell lysates by ion-exchange chromatography. In the fraction that showed maximal IgM binding, the dominant protein was identified as the 47-kDa translational elongation factor 1alpha (eEF1alpha). We used the donor's B-cells to create hybridomas producing the antibody (B12.6.22) that bound to neuroblastoma cells and mediated cytotoxicity. This antibody recognized eEF1alpha in a specific manner. Sequence analysis of the heavy chain of B12.6.22 showed usage of VH3-23 and JH6 gene segments, with no somatic mutation. The structural similarity of B12.6.22 to antibodies of the innate immune system supports the assumption that natural antibodies are a potential source of therapeutic antibodies.

Sea urchin elongation factor 1delta (EF1delta) and evidence for cell cycle-directed localization changes of a sub-fraction of the protein at M phase.

Eukaryotic elongation factor 1 (eEF1) is a translational multimolecular complex reported in higher eukaryotes to be a target of CDK1/cyclin B, the universal regulator of M phase, but whose role in the cell cycle remains to be determined. A specific polyclonal antibody was produced and used to characterize the delta subunit of sea urchin elongation factor 1 (SgEF1delta) in early embryos, a powerful model for investigating cell cycle regulation. The SgEF1delta protein was present in unfertilized eggs as two isoforms of 35 and 37 kDa, issued from two different mRNAs. The two canonical eEF1delta partners, eEF1gamma and eEF1beta, were shown to co-immunoprecipitate with the SgEF1delta isoforms. Both isoforms were associated in a macromolecular complex, which resolved upon gel filtration chromatography at a molecular weight > 400 kDa, suggesting association with other yet unidentified partners. After fertilization, the amount as well as the ratio of both SgEF1delta isoforms remained constant during the first cell division as judged by Western blotting. Immunofluorescence analysis showed that a pool of the protein concentrated as a ring at the embryo nuclear location around the period of nuclear envelope breakdown and was visualized later as two large spheres around the mitotic spindle poles. Thus, the eEF1delta protein shows cell cycle-specific localization changes in sea urchin embryos.

Monoclonal antibodies to elongation factor-1alpha inhibit in vitro translation in lysates of Sf21 cells.

Elongation factor-1alpha (EF-1alpha) is an enzyme that is essential for protein synthesis. Although EF-1alpha offers an excellent target for the disruption of insect metabolism, agents known to interfere with EF-1alpha activity are toxic to humans. In this article, we describe the development of monoclonal antibodies (MAbs) that can disrupt the activity of insect EF-1alpha without cross-reacting with the human enzyme. MAbs were generated to EF-1alpha from Sf21 cells derived from the fall armyworm, Spodoptera frugiperda, by immunizing mice with EF-1alpha eluted from SDS-PAGE gels. The MAbs reacted with EF-1alpha in eggs and first through fifth instars of the fall armyworm in immunoblots of SDS-PAGE gels, but did not recognize EF-1alpha in human carcinoma cells and normal tissues. MAbs with the ability to recognize EF-1alpha in its native conformation, identified through immunoprecipitation experiments, were added to Sf21 cell lysates to determine whether the antibodies could inhibit incorporation of [(35)S]methionine into newly synthesized in vitro translation products. Of the four EF-1alpha-specific MAbs tested, three significantly inhibited protein synthesis when compared to the negative control antibody (P < 0.001, one-way ANOVA; followed by Dunnett's test, P < 0.05).

Elongation factor 1beta is an actin-binding protein.

A 17 kDa polypeptide found in association with actin in cellular extracts of Dictyostelium discoideum was identified as a proteolytic fragment of eEF1beta. Antibody elicited against the 17 kDa protein reacted with a single 29 kDa polypeptide in Dictyostelium, indicating that the 17 kDa peptide arises from degradation of a larger precursor. The cDNA isolated from a Dictyostelium library using this antibody as a probe encodes Dictyostelium elongation factor 1beta. Amino acid degradation of the 17 kDa protein fragment confirmed the identity of the protein as eEF1beta. Direct interaction of eEF1beta with actin in vitro was further demonstrated in mixtures of actin with the 17 kDa protein fragment of Dictyostelium eEF1beta, recombinant preparations of Dictyostelium eEF1beta expressed in Escherichia coli, and the intact eEF1betagamma complex purified from wheat germ. Localization of eEF1beta in Dictyostelium by immunofluorescence microscopy reveals both diffuse cytoplasmic staining, and some concentration in the cortical and hyaline cytoplasm. The results support the existence of physical and functional interactions of the translation apparatus with the cytoskeleton, and suggest that eEF1beta may function in a dual role both to promote the elongation phase of protein synthesis, and to interact with cytoplasmic actin.